Resolving the 3D spatial orientation of helix I in the closed state of the colicin E1 channel domain by FRET. Insights into the integration mechanism

Archives of Biochemistry and Biophysics 608 (2016) 52e73

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Resolving the 3D spatial orientation of helix I in the closed state of the colicin E1 channel domain by FRET. Insights into the integration mechanism Miguel R. Lugo, Derek Ho, A. Rod Merrill* Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 June 2016 Received in revised form 27 July 2016 Accepted 8 August 2016 Available online 3 September 2016

Current evidence suggests that the closed-state membrane model for the channel-forming domain of colicin E1 involves eight amphipathic a-helices (helices IeVII and X) that adopt a two-dimensional arrangement on the membrane surface. Two central hydrophobic a-helices in colicin E1 (VIII and IX) adopt a transmembrane locationethe umbrella model. Helices I and II have been shown to participate in the channel by forming a transmembrane segment (TM1) in the voltage-induced open channel state. Consequently, it is paramount to determine the relative location and orientation of helix I in the twodimensional arrangement of the membrane. A new, low-resolution, three-dimensional model of the closed state of the colicin E1 channel was constructed based on FRET measurements between three naturally occurring Trp residues and three sites in helix I, in addition to previously reported FRET distances for the channel domain. Furthermore, a new mechanism for the channel integration process involving the transition of the soluble to membrane-bound form is presented based on a plethora of kinetic data for this process. Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.

Keywords: Colicin E1 FRET Lifetime fluorescence Molecular modeling Ion channels

1. Introduction Colicins are antimicrobial proteins produced by Escherichia coli cells that target susceptible bacteria in response to stressful conditions, including nutrient depletion, DNA damage, overcrowding and anaerobiosis [1]. Colicins can be grouped into three major categories based on their routes of lethal action: (i) the formation of a depolarizing ion channel in the cytoplasmic membrane; (ii) the inhibition of protein and peptidoglycan synthesis, and (iii) the degradation of nucleic acids [2]. Colicin E1 (ColE1) is a member of the ion-channel forming group of colicins which also includes colicins A, B, Ia, Ib, K, and N [3,4]. The C-terminal channel-forming domain of ColE1, ColE1c, forms a dissipative ion channel which depolarizes the cytoplasmic membrane of target bacterial cells [5]. Before insertion into the membrane, the ColE1c peptide undergoes a series of structural changes by first binding to the lipid bilayer, followed by protein unfolding and helix elongation [6e8]. The crystal structure of the soluble channel domain (2.5 Å) [9,10] is

* Corresponding author. Department of Molecular and Cellular Biology, Science Complex, University of Guelph, Guelph, ON, N1G2W1, Canada. E-mail address: [email protected] (A.R. Merrill). http://dx.doi.org/10.1016/j.abb.2016.08.007 0003-9861/Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.

comprised of 10 a-helices that form an extremely stable, watersoluble globular protein [11,12]. Interestingly, this protein consists of a hydrophobic a-helical hairpin, helices VIII and IX (H8 and H9), which acts as the non-polar core of the protein and becomes transmembrane upon membrane association [13]. These two helices are critical to colicin pore formation as they create a membranespanning hairpin that anchors the channel within the bilayer [14e16]. The additional 8 a-helices of the channel peptide were shown to be amphipathic elements that surround the hydrophobic core of the channel [17e20]. In the membrane environment, the channel peptide forms a structure that has been described as an umbrella model in which only the hydrophobic helices, H8 and H9, are inserted into the nonpolar core of the membrane, with the amphipathic helices splayed out onto the membrane surface [21,22]. The channel then opens in response to a trans-negative membrane potential, facilitating the escape of various ions from the host cells, such as Naþ, Kþ, and Hþ, leading to host cell death [23]. The umbrella model has received strong experimental support from time-resolved FRET studies of ColE1c [6,24,25]. It has been shown that the eight amphipathic ColE1c a-helices on the membrane surface adopt a two-dimensional arrangement [26] and FRET

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2. Material and methods Abbreviations ANM CD ColE1 DMG DW EHT ESM FRET GNM Gu-HCl IMAC LUV mBBr P190H6 SASA SC SVL TM TNP WT

anisotropic network model circular dichroism colicin E1 dimethylglutamic acid Durbin-Watson statistics extended Hückel theory exponential series method fluorescence resonance energy transfer Gaussian network model guanidine hydrochloride immobilized metal affinity chromatography large unilamellar vesicles monobromobimane 190-residue channel domain of colicin E1 with an N-terminal hexahistidine tag solvent-accessible surface area spectral centroid scientific vector language trans-membrane trinitrophenyl wild-type

data for colicin A revealed that distances generally increased upon membrane association [27e29]. However, the exact orientation of the helices as well as the details of the lipid and protein contacts is still poorly understood. A previous study involved FRET analysis where Cys505 was labeled with I-AEDANS as an acceptor and 11 Trp donor residues were randomly situated throughout the channel domain. These results revealed that higher relative changes in FRET efficiencies were observed the closer the Trp donor was to the Nterminus of the protein [24]. In support of the umbrella model, Cramer and coworkers adopted a similar approach by using FRET to probe the relative distance of each helix to Cys509 [26]. Although the data could be accounted for by the formation of a quasi-circular arrangement of the eight amphipathic a-helices laying on the membrane surface, a number of other models with various twodimensional configurations of the helices are also possible. To further test the proposed quasi-circular arrangement model, our lab used the system developed by Schultz [30] to incorporate coumarin into the ColE1 channel domain to act as an intrinsic FRET donor with DABMI-labeled Cys residues as the acceptor [31]. This approach led to the development of a circular arrangement model with helices IeVII (H1eH7) arranged in a clockwise direction from the extracellular side around the central transmembrane hairpin formed by H8 and H9 [32]. Helices I and II (H1 and H2) have been shown to participate in the translocation pore by forming a transmembrane segment (TM1) in the voltage-induced open-state [22]. Hence, it is important to further refine and/or update our proposed model, with special emphasis in the relative location and orientation of H1eH2. This study involves the construction of a new, low-resolution 3Dmodel of the closed-state ColE1 channel-domain based on FRET measurements between three endogenous Trp residues, and three positions in H1, while taking into account most of the published FRET distances [26,33]. In addition, we revise and give a new interpretation of published kinetic data of the integration process from the soluble to the membrane-bound form [26,34].

2.1. Preparation of single-Trp, single-Cys ColE1 variants A total of 12 single-Trp and single-Cys variants were prepared using the P190H6 construct with the C505A mutation by sitedirected mutagenesis as previously described [33]. All plasmids were purified using the High Pure Plasmid™ isolation kit from Roche Diagnostics (Laval, PQ, Canada) and mutation sites were confirmed by DNA sequencing (Univ. of Guelph). Two groups of variants were prepared for the FRET studies. The first group comprised three-distinct, single-Trp and single-Cys variants specifically designed for FRET distance measurements between the three intrinsic Trp sites (Fig. 1). For each variant, one of the three naturally occurring Trp residues (Trp424, Trp460 and Trp495) was replaced with Phe to remove the fluorescent signal, and the other with a Cys residue to act as an acceptor labelling site, according to the distance to measure by: (1) W424C/W495F for the Bim424eTrp460 distance, (2) W495C/W424F for Bim495eTrp460, and (3) W424C/W460F for Bim424eTrp495. The second group comprised nine single-Trp and single-Cys variants for the purpose of FRET distance measurements between H1 and the three naturally occurring Trp residue sites. Three residues (Asp347, Ser354, Glu361) within H1 were selected for Cys replacement. These residues were selected, firstly, because they are equidistant and throughout the length of H1; and secondly, because they are exposed to the solvent, which should reduce the impact on the expression and folding of the polypeptide, and on the structure and stability of the purified protein. In each variant, two of the three intrinsic Trp residues were replaced with Phe residues, while one of the three selected H1 residues was replaced with Cys to act as an acceptor labelling site, according to: (1) D347C/W460F/W495F for the Bim347eTrp424 distance, (2) D347C/W424F/W495F for Bim347eTrp460, (3) D347C/W424F/ W460F for the Bim347eTrp495 distance, (4) S354C/W460F/W495F for Bim354eTrp424, (5) S354C/W424F/W495F for the Bim354eTrp460 distance, (6) S354C/W424F/W460F for Bim354eTrp495, (7) D361C/W460F/W495F for the Bim361eTrp424 distance, (8) D361C/W424F/W495F for Bim361eTrp460, and (9) D361C/W424F/W460F for Bim361eTrp495. In this study, the lone Cys residue (Cys505) in all 12 variants was replaced with Ala to avoid the possibility of non-specific labelling. Previous studies confirmed that the C505A mutation does not perturb either the secondary or tertiary structure of the channel domain [33]. However, for the sake of simplicity, the C505A notation is omitted in the variant nomenclature.

2.2. Protein purification and monobromobimane Both the wild-type (WT) P190H6 and the single-Trp single-Cys variants were prepared from transformed lex A Escherichia coli IT3661 cells and purified using Immobilized Metal-Affinity Chromatography (IMAC) as previously described [33]. Protein purities were assessed by SDS-PAGE and protein concentrations were determined by spectroscopy at A280, using an extinction coefficient (ε) of 29910 M1 cm1 for WT protein (ε ¼ 17210 M1 cm1 for single Trp variant protein) [35]. Purified single-Trp, single-Cys variants were labeled with the small fluorophore monobromobimane (mBBr, 271.11 g/mol) (Molecular Probes, Eugene, USA) at a 20:1 M ratio (probe:protein), and the labeling efficiency was determined as previously described [32].

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Fig. 1. Schematic representation of the colicin E1 channel domain. (A) Cartoon representation of the 12 intra-molecular distances measured by FRET in this study using the ribbon topology diagram of the 2.5 Å crystal structure of the P190 peptide (PDB: 2i88). The three naturally occurring Trp residues: Trp424 (blue), Trp460 (red) and Trp495 (green) are depicted in stick format, while bimane at the three selected positions (347, 354 and 361) in helix I (H1, in red ribbon) are represented by 2D sketches. The lines connecting the molecular center of Trp and Bim are matched according to the Trp color. Inter-Trp distances are shown in black lines. (B) Primary sequence and secondary structure of ColE1c. The colored residues were subjected to either Cys (cyan) or Phe codons replacement for the purpose of this study. The naturally occurring Cys505 was replaced with Ala505 in all variants. The color of the expressed Trp residue corresponds to the one in the upper panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.3. Preparation of large unilamellar vesicles (LUVs)

2.5. Fluorescence lifetime measurements and data analysis

LUVs were prepared from 1,2-dioleoyl-sn-glycero-3phosphocholine and 1,2-dioleoyl-sn-glyerco-3-[phospho-rac-(1glycerol)] vesicles at a 60:40 M ratio (Avanti Polar Lipids). Lipids were prepared and quantified as described previously [19], except the buffer used to suspend vesicles consisted of 10 mM DMG and 100 mM NaCl (pH 4.0). Asolectin (Fluka) was purified according to the method of Schendel and Reid [36], and vesicles were prepared as described previously [37]. Phospholipid concentration was determined using the microBartlett assay [19].

Pulsed-fluorimetry was carried out on a PTI (Photon Technology International Inc., London, CA) TimeMaster Model TM4 system, employing a low repetition rate pulsed nitrogen laser (at 10 Hz with 0.6 ns half-width pulses) pumping a PTI GL-302 high-resolution dye laser. The dye laser output at 590 nm was frequency-doubled to 295 nm, and the emission was observed at 90 relative to the excitation. Since the excitation light is virtually unpolarized, emission was detected through a single polarizer set at 35 . The emission was collected for 40 ns at 340 nm, by using a photomultiplier stroboscopically controlled [38] in a cell holder thermostated at 22  C. The expected pulse response of the sample, E(t), is the convolution of the fluorescence decay function, F(t), with the pulse response of the instrument, I(t), as given by

2.4. Absorbance and fluorescence emission spectra measurements The absorbance of the mBBr-Cys labeled variants were measured as previously described [19] in a Helma ultramicro absorbance cuvette (light path 10 mm) using a Cary 300 spectrophotometer, scan range 250e650 nm. Fluorescence emission of the Trp residues was measured at an excitation wavelength of 295 nm and emission was scanned between 305 and 460 nm with 1 nm steps and 0.2 s integration time. Excitation and emission bandpasses were set at 4 and 8 nm, respectively.

Zt EðtÞ ¼ IðtÞ5FðtÞ ¼

IðkÞFðt  kÞdk

(1)

∞

I(t) was recorded at the excitation wavelength, using a scattering solution of colloidal silica (LUDOX) at 0.0005% as sample. The decay

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function was assumed to be multi-exponential, as defined by

FðtÞ ¼

n X

t

ai $e ti

(2)

1

where n is the total number of components of the decay, and ai and ti are the fluorescence intensities at time zero and the average lifetime of the ith component, respectively. The function E(t) was fit to the observed decay, O(t), by two approaches for the F(t) function: (i) a discrete lifetime analysis with up to 4 exponential components, and (ii) a lifetime distribution analysis featuring the exponential series method [38] ESM, with 200 components logarithmically spaced in the range from 0.01 to 8 ns. Both methods were iteratively performed to guide one to the other until (a) both converged in a reduced c2 around 1.0, (b) the distribution centers match the values reported by the discrete analysis, (c) and likewise the fractional areas (intensities) assigned for each component. During the fitting by the discrete analysis, in some cases, a missing component (usually a fast decay with low amplitude) was revealed by the lifetime analysis. The further inclusion of this component (either fixed or free-floating) in the discrete analysis significantly improved the quality of the fit. Reciprocally, the output from the discrete analysis sets the offset to pre-process the raw decay, and defines the search range for the distribution analysis. In addition, residual graphics, Durbin-Watson statistics and autocorrelation curves were used to assess the quality of each fit. The amplitudeweighted average was calculated by

P i ai ti 〈t〉 ¼ P

a

2.6. Calculation of FRET efficiency and FRET distance The efficiency (E) of the resonance energy transfer, FRET, is related to the inverse sixth power of the distance (R) between a “donor” (D) and an “acceptor” (A) molecules by

(4)

€rster distance) is the distance (in Å) at which the where R0 (the Fo efficiency of energy transfer is 50%. R0 can be calculated by using the equation

1  6 R0 ¼ 0:211 J$QD $k2 $n4

k2max in the 1.8e2.4 range, depending on the donor reorientational freedom [39], producing a maximum variation of the calculated distance between 13% and þ23% with respect to the ‘2/3’ distance. The FRET efficiency E can be obtained experimentally by either measuring the intensity (steady-state) or the lifetime (timeresolved) fluorescence of the donor in the presence and absence of the acceptor, as described by

E ¼1

FDA t ¼ 1  DA FD tD

(7)

where FD and FDA represent the steady-state intensities, while tD and tDA represent the lifetime averages of the donor fluorescence in the absence and presence of the acceptor (Eq. (3)), respectively. 2.7. Protein thermal melts The melting scan of each single-Trp variant (5 mM in buffer at pH 6.5), was conducted in the presence of 0.5 M Gu-HCl to increase the solubility and was performed in a PTI Quanta Master 300 steadystate fluorimeter, with the cell holder Peltier-based thermostated with a thermal ramp changing from 5  C to 80  C at a rate of 0.25  C/ min, and magnetic stirring. The system was set to automatically record the emission spectrum at 5  C increments between in the range of 315e450 nm (lex ¼ 295 nm), at the rate of 300 nm/min (1 nm steps). The denaturation of the protein was monitored by calculating the spectral centroid (SC) of each emission spectrum by

Z (3)

which uses normalized amplitudes for weighting the time constant (ti) of each decay component.

 1 6 R ¼ R0 E1  1

55

SC ¼ Z

l$FðlÞdl (8) FðlÞdl

where F(l) is the blank-corrected intensity of the fluorescence at the wavelength l. The melting curve was fit with OriginPro 8.0 (OriginLab Corp. MA, USA) to an adapted sigmoidal expression [40], by

SCðTÞ ¼ ðSCU þ mU $TÞ þ

ðSCF þ mF $TÞ  ðSCU þ mU $TÞ ðTTm Þ dT

1þe

(9)

where the subscript U and F makes references to the unfolded and folded protein species, respectively. The coefficient, mi, characterizing the linear temperature-dependent fluorescence of each species; SCF and SCU are the spectral centroid of each species at 5 and 80  C, respectively; Tm is the transition temperature; and dT is the transition slope.

(5) 2.8. Molecular modeling

where QD is the fluorescence quantum yield of the donor, n is the refractive index of the medium, k2 is the orientation factor, and J is the spectral overlap integral (in M1 cm1 nm4), between the donor emission and acceptor absorbance spectra, was calculated by

Z J¼

FD ðlÞ$3 A ðlÞ$l4 dl Z FD ðlÞdl

(6)

where FD(l) is the fluorescence emission spectrum of the donor alone, and 3 A ðlÞ (in M1 cm1) is the molar extinction coefficient of the acceptor. The orientation factor was assumed to be k2 ¼ 2/3, €rster distance. This which is the default for the calculation of the Fo value is compatible with anisotropy measurements for the bimane probes [19], which limits k2 to a k2min in the 0.3e0.5 range, and a

For the molecular modeling and rendering, the Molecular Operative Environment, MOE 2015 software package (Chemical Computing Group, Montreal, CA) was used. For the “soluble-form” of the channel domain, the PDB:2i88 X-ray structure was used, while for the “membrane-form” a template consisting of an embedded model was employed as initial coordinates [33]. For the in silico labelling of both forms of the protein, the final adducts were optimized in an implicit solvent model using the Amber10:EHT force-field. For the mapping strategy, firstly a “manual” stage was performed by rigid-body manipulations of the individuals helices and hairpins by in-house routines programmed with the MOE scientific vector language (SVL) compiler. For a further “energyminimization” stage, to the MOE energy potential function, a restraint energy term, Eres, was incorporated to set the FRET distances as distance-constraints by

56

E¼

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X

EFF þ Gsol þ

X

Eres ðdÞ

(10)

with EFF being the standard bonded and non-bonded force-field terms, and Gsol the solvation energy. Each constraint penalizes any deviation of the actual inter-probe distance, d, from a reference value do (the FRET distance), according to a lower (L ¼ do  d) and an upper (U ¼ do þ d) thresholds by

Eres ðdÞ ¼ w½pðL  dÞ þ pðd  UÞ

(11)

with w being an arbitrary force constant, and p a MOE built-in function, which is 0 when evaluated inside the range [L,U], but increases with the absolute value of the difference, jd  do j; when evaluated out of the range. The d variation was arbitrary set to 2 Å. A search routine for optimizing side-chain atoms and loop structures was performed by using the MOE conformational search protocol LowModeMD [41]. The algorithm was subjected to 10,000 iterations or terminated sooner (after 100 iterations) if it continually failed to produce a new conformation. A conformation was considered new if after the energy minimization step (to the RMS gradient of 0.01 kcal/mol/Å2) it was within a defined energy window of 20 kcal/mol from the lowest structure in the database, and it was not duplicated according an RMSD threshold of 0.25 Å. 2.9. Modeling strategy for the mapping of the membrane-bound form of ColE1c Table 3 presents three sets of FRET distances used for the mapping. The first set corresponds to ‘inter-helices’ distances, obtained by six DABMI/Coumarin FRET pairs that measure the distance between the first six contiguous helices [33]. The second set corresponds to ‘509eradial’ distances between five Trp/AEDANS FRET pairs [26] having in common the labeling of AEDANS (FRETacceptor) at the 509 site, located in the L9/10 loop. In this set, the FRET-donors are three variants (Trp356, Trp396 and Trp413) and two naturally occurring (Trp424 and Trp460) Trp residues, located throughout the external helices (H1eH7) and connecting loops. The third set corresponds to the ‘TrpeH1’ distances reported in this work, which include nine Trp/Bim FRET pairs obtained by permuting three bimane FRET-acceptors located in H1 with the three naturally occurring Trp FRET-donors (Trp424, Trp460 and Trp495). The modeling strategy implemented in this study presents several improvements in relation to our previous work [33]. First, it was possible to simultaneously use the larger set of FRET distances by synthesizing ein silicoe a multi-adduct protein with all the probes herein considered (Trp, bimane, AEDANS, DABMI, and coumarin), to reproduceein the same protein moleculeeall the experimental FRET pairs. To achieve this, the experimental labeling of the Cou390/DABMI411 and Cou426/DABMI441 FRET pairs [26] were swapped to constitute a series of FRET pairs connecting contiguously the first six helices by alternating the DABMI and the coumarin probes (3rd and 5th pairs, Table 3); ensuring that each labeling occurs at a particular residue. Second, rather than using the inter Cb-atom coordinates for the mapping, in the new model, “center” coordinates were assigned to the actual probes. For some probes, their center is located quite distant from the anchor point at the Ca-atom. This is the case for the AEDANS and DABMI dyes, which are elongated molecules with main molecular axes as long as ~10 and 14 Å, respectively, in their extended conformations. The improvement resulting from the inclusion of the actual dimensions and the center of the probes is evident in the better agreement between the center-to-center and the FRET distances for the soluble ColE1c (see Results). Third, a further improvement in the

modeling strategy was implemented by using the FRET distances as energy-constraints in an energy minimization protocol (Eqs. (10e11)). In this aspect, a constant grade of flexibility was allowed in the mobility of the probes by defining a range of ±2 Å around the reference value, do, without an energy cost. Out of this range, the energy restraint constant that penalizes the deviation was set to a low value to avoid overriding force-field energy components and, therefore achieving an unnatural geometry (i.e., bond lengths, dihedrals, etc.). The first stage of the mapping protocol, step (i), was primarily based on geometric criteria. Each helix or hairpin of the labeled protein was taken independently as a solid-body and positioned onto/into the membrane according to the depth and tilt angle [33] (Table S1). Thus, the H8/9 hairpin was positioned exactly in the location and orientation exhibited in the Ho model. However, the H1/2 and H6/7 hairpins, and the rest of the individual a-helices (H3eH5, and H10) were manually positioned and/or rotated in the XY-plane in order to simultaneously match (roughly, within a variation of ±10 Å) the complete set of FRET distances. Details concerning the underlying reasoning that guided this initial position is presented in Supplementary Materials. A further modeling stage was conducted by: (ii) fixing the coordinates of a-helices backbone atoms; (iii) geometry optimization of side-chain atoms and all atoms in random loops by FRET-restricted energy minimization, in order to reconfigure the loops and correct side-chain conformations; (iv) assessment of the loop backbone geometry, bonded (strain) energy of the side-chains, restraint energy of the FRET pairs, as also non-bonded interactions (mainly H-bonds and van der Waals energies); (v) unfixing of all a-helix backbone atoms except for the hairpin H9/10; (vi) relaxation of any remaining atom clashes and restraint energies by manual rigid-body XY-rotation/ translation of any a-helix or hairpin according to the energy evaluation of the step (iv); and (vii) a conformational search over sidechain and loop backbone conformations by using the LowModeMD methodology described above. The steps (ii) to (vii) were iterated with a progressively decreasing restraint force constant (Eq. (11)) in each cycle (from w ¼ 5.0 to 0.5 kcal/mol/Å2), until a 2D-arrangement of external helices arranged on the membrane was obtained that fulfilled both the previous and the new FRET distances. In addition, the model must also satisfy protein geometry (backbone dihedrals, side-chain torsionals, bond angles and bond lengths), and low or no atom clashes. This mapping protocol was performed first with the 20 distances of the “modeling set” reported in Table 3. However, it was noted that with the inclusion of the 5th (DABMI426/Cou441) and 10th (AEDANS509/Trp424) pairs, the refined model presented a high average deviation with the FRET distances, high restraint energy, and a low “quality” geometry according to the large number of outliers in bond angles and bond lengths (see Results for further details). 2.10. Kinetic modeling The kinetic scheme, synthetic time-course observables, and fitting curves (Eqs. (12e20), Results section) were solved with the software KinTek Global Kinetic Explorer (KinTek Corp., Snow Shoe, USA). 2.11. Structural dynamics analysis Atomic fluctuations by the Gaussian Network Model (GNM), and the Anisotropic Network Model (ANM) were performed in the Protein Dynamics (ProDy) package release 1.7 [59] running on Python 2.7.9 (Python Software Foundation, Delaware, USA). Details on the theoretical and practical aspects of the GNM and ANM methods

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were reported earlier [42]. 3. Results 3.1. Protein expression and purification All single-Trp and single-Cys variants were expressed and purified as previously described [33]. The purity of the proteins was assessed by SDS-PAGE analysis with 95% homogeneity or higher. All single-Trp and single-Cys variants showed WT expression levels except for W460C/W495F (for the Bim460eTrp424 distance determination by FRET), which showed no colicin expression. To resolve this issue, the variant W424C/W495F was prepared by switching the donor/acceptor position, i.e., Trp460eBim424. This new variant shown WT expression levels, suggesting that Trp460 is a critical residue for the overall structural integrity. However, the position 460 tolerates substitution with a Phe residue (e.g. D347C/ W424F/W460F), so the phenyl ring seems to be a minimal chemical requirement to maintain the folding of the channel peptide. Incidentally, the double-variant protein corresponding to the single emitter Trp460 (W424F/W495F) is more stable than the equivalents for Trp424 (W460F/W495F) and Trp495 (W424F/W460F), according to their unfolding transition temperatures (70.0, 63.3 and 61.3  C, respectively) monitored by changes in the spectral centroid of the emission spectra upon thermal denaturation (Fig. S1). 3.2. Spectroscopic measurement of Trp and mBBr-Cys tethered adducts To assess the chemical properties of the bimane-labeled and non-labeled ColE1c variants, both the absorbance and fluorescence emission spectra were collected (Fig. S2). The absorbance spectra demonstrated the successful formation of the mBBr-Cys tethered adduct with the presence of an absorbance peak near 380 nm. The fluorescence emission spectra, with maxima around 320ee340 nm, correspond to the Trp emission. Subtle changes were observed for the emission spectra upon membrane association. In general, the intensity of Trp emission decreased upon the addition of LUVs. In contrast, the absorbance spectra remained €rster distance (R0) was unchanged upon membrane-binding. The Fo calculated based on the overlap integral between the emission and absorbance spectra (see below). 3.3. Time-resolved fluorescence measurements for the soluble state of ColE1c The soluble ColE1c protein has been well characterized both structurally [9], and spectroscopically [22,24,25,43,44]. Herein, the observed decays of the intrinsic fluorescence of the three single-Trp residues of both unlabelled and bimane-labeled Cys-variants at pH 7.0, were analyzed by two fitting approaches (see Material and Methods). For example, Fig. 2 shows the convoluted fluorescence decay and the lifetime distributions for the Trp424 signal in the variant E347C (Trp424/Cys347). The shaded rows in Table 1 correspond to the fluorescence emission of the three single Trp emitters (“donors” of the FRET pair), each with the three Cys-variants at H1. The average lifetimes correspond with WT values reported earlier [43] and are inversely correlated with the degree of exposure to the solvent, as has been reported for acrylamide quenching assays [45] and by computational evaluation of the SASA in molecular dynamics simulations [46]. For the bimane-labeled Cys-variants, clear rows in Table 1, the Trp average lifetimes are lower than the respective unlabelled ones, in agreement with the role of bimane as the “acceptor” molecule in a FRET pair. Notably, in some cases, the number of decay

Fig. 2. Fitting of the fluorescence decay of the Trp424/Cys347 variant of ColE1c. (A) A set of 400 reading (dots) of the convoluted Trp signal fit to a bi-exponential function. The time constant of each component, t1 and t2, yielded an amplitude-weighted average of 2.99 ns. (B) Bimodal lifetime distribution from the fit of the convoluted signal in the upper panel to 200 logarithmically-spaced exponential decays. The peaks correspond closely to the lifetime constant reported by the discrete analysis, and the relative areas (Ai) to the fractional intensities (fi).

components increased with respect to the unlabelled ones, and for some individual components their tDA are higher than tD. For example, the fastest “detected” component of the Trp460/Bim347 FRET pair has a longer lifetime than in its unlabelled counterpartethe Trp460/Cys347 variant. The same phenomenon happens in the intermediate component (t2) of the Trp495/Bim347 FRET pair, in comparison with its counterpart in the unlabelled Trp495/ Cys347 variant. This led us to establish the hypothesis that each component of the original Trp decay would be quenched differently by FRET in two signals due to the bimane conformational distribution. Molecular dynamics simulations of an in silico adduct of bimane at the exposed 473 residue (at L7/8), showed correlated transitions among its c2, c3 and c4 dihedrals that yielded two stable bimane conformers (unpublished data). The previous transition is evident in the Trp460 signal with/without labelling at residues 354 and 361, where the mono-exponential decay for the donor-alone (just one Trp conformer) becomes bi-exponential in the presence of the acceptor bimane (with two conformers). Accordingly, our working hypothesis establishes that the shortest component of the Trp460/Bim347 signal (t2,DA), for example, has its origin from the

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Table 1 Fluorescence lifetime measurements of the unlabeled and bimane-labeled single-Trp ColE1c. Time constants (t, ns) and relative intensity (f) of each component of a multiexponential fitting of the fluorescence decay signal from the Trp donor alone (shaded rows) and donor-acceptor pair (clear rows) for the soluble ColE1c at pH 7.0 (upper panel), and for the membrane-bound form at pH 4.0 (lower panel). The corresponds to amplitude weighted average lifetime (eq. (3)), c2 and DW to the reduced chi-square and the Durbin-Watson statistics, respectively. Signal

Unlabelled labeled

Lifetime (fractional intensity)

Statistics

t1 (f1)

t2 (f2)

t3 (f3)



c2

DW

0.12 (0.02)

1.34 (0.24) 0.66 (0.22)

4.84 (0.76) 3.46 (0.76)

2.99 1.40

0.97 1.08

1.78 2.24

1.39 (0.49)

5.01 (0.51)

2.20

1.07

2.18

Bim354

0.23 (0.02)

0.68 (0.31)

3.23 (0.67)

1.32

1.22

1.75

Cys361 Bim361

0.39 (0.04) 0.32 (0.12)

1.96 (0.11) 0.81 (0.33)

4.84 (0.85) 3.11 (0.55)

2.88 1.04

1.00 1.07

1.63 1.95

Cys347

0.42 (0.02)

4.51 (0.98)

3.66

0.98

2.03

Bim347

1.00 (0.07)

4.24 (0.93)

3.50

0.96

1.97

4.56 (1.00)

4.56

1.00

2.25

0.53 (0.02)

3.84 (0.98)

3.48

0.85

2.23

4.36 (1.00)

4.36

1.00

2.26

0.33 (0.02)

3.87 (0.98)

3.11

1.16

1.91

tο (fo) Soluble form at pH 7.0 Trp424

Cys347 Bim347 Cys354

Trp460

Cys354 Bim354 Cys361 Bim361 Trp495

Cys347 Bim347

0.24 (0.03)

2.11 2.53

1.20 (0.02)

4.65 (0.98)

4.35

1.08

2.29

3.63 (0.98)

3.34

1.05

1.90

Cys347

<0.05 (0.07)

1.37 (0.07)

4.54 (0.93)

3.94

1.08

1.68

0.76 (0.07)

3.79 (0.93)

3.00

0.99

1.93

1.23 (0.13)

3.30 (0.55)

7.23 (0.25)

1.29

1.01

2.35

0.11 (0.16)

0.82 (0.26)

3.51 (0.58)

0.50

0.96

2.27

0.77 (0.18)

4.04 (0.82)

2.31

0.99

2.13

Bim354

0.18 (0.21)

0.87 (0.31)

2.96 (0.48)

0.60

0.98

1.72

Cys361

0.76 (0.21)

Cys354

Trp460

1.02 0.92

0.70 (0.02)

Bim347

Trp495

2.77 2.60

Cys354 Cys361

Trp460

4.54 (0.89) 4.30 (0.90)

Bim354 Bim361 Membrane-bound Form at pH 4.0 Trp424

0.68 (0.11) 1.61 (0.07)

2.85 (0.24)

4.63 (0.55)

2.70

0.84

1.78

Bim361

0.68 (0.13)

2.96 (0.56)

1.20

1.27

2.27

Cys347

0.09 (0.02)

4.35 (0.98)

2.17

1.24

1.42

Bim347

0.10 (0.05)

4.01 (0.95)

1.38

1.07

1.64

Cys354 Bim354

0.93 (0.03) 1.37 (0.04)

4.11 (0.97) 3.86 (0.96)

3.72 3.62

0.94 1.15

1.76 2.09

Cys361

0.11 (0.03)

4.02 (0.97)

2.05

1.01

1.84

Bim361

0.13 (0.04)

3.80 (0.96)

1.90

1.04

2.10

Cys347 Bim347

1.64 (0.14) 0.27 (0.09)

5.07 (0.86) 4.14 (0.91)

3.91 1.81

1.19 1.01

1.41 1.89

Cys354

0.41 (0.03)

4.45 (0.97)

3.45

1.07

2.11

Bim354

0.85 (0.03)

3.54 (0.97)

3.28

1.54

1.80

Cys361 Bim361

0.30 (0.03) 0.33 (0.06)

4.32 (0.97) 3.76 (0.93)

3.12 2.26

1.03 0.94

2.09 1.91

Cys424

0.54 (0.02)

4.12 (0.98)

4.05

1.35

1.70

1.85 (1.00)

1.85

1.02

1.93

4.05 3.04

1.01 0.95

1.92 2.09

Bim424 Trp460

Cys495 Bim495

3.51 (0.72)

6.85 (0.27) 3.04 (1.00)

Trp495

Cys424

2.67 (0.28)

4.92 (0.71)

3.97

1.22

1.78

Bim424

0.77 (0.39)

2.75 (0.60)

1.37

1.06

2.09

Bold value indicates the average lifetime for each variant.

longest (t3,D) component of the Trp460/Cys347 signal, rather than from its shortest component (t2,D), explaining the higher t2,DA than t2,D values. This hypothesis requires, in principle, that both probes explore their conformational spaces independently, as it illustrated in Fig. 3. Exploratory adiabatic maps for the Trp conformers have revealed more than one minimum in the c1ec2 plane, separated by large energy barriers [46]. In this sense, a strong correlation between the Trp rotamer distributions and the fractional amplitudes of fluorescence lifetimes were observed [47], even for the naturally occurring Trp in colicin A [48]. However, the reduced number of “detected” decay components for the donor-acceptor pairs (no more than 3 components, Table 1) in comparison to the expected number (up to 6 components by considering all the possible

combinations between states/conformers of the Trp and Bim residues) by splitting each donor-alone component, can be accounted for by the increased deactivation of the Trp signal by bimane and/or the low population for some combinations of the Trp and Bim conformers. Thus, the faster donor-alone decay components (t1,D and t2,D) might not be observed in the deconvoluted donoracceptor signals either for their short lifetimes (tDA < 0.05 ns) and/or for their low intensities (scarce populations, ai ≪ 1). This might explain why the t1,DA component only appears in the cases where its source signal, t2,D, is relatively populated (i.e., f2,D > 0.1). In some cases with multi-exponential decays for the donor-alone emission (e.g., all the Trp424 signals), t2,DA is indeed lower than t2,D, partially obscuring the splitting phenomenon by FRET

M.R. Lugo et al. / Archives of Biochemistry and Biophysics 608 (2016) 52e73

59

Fig. 3. Model of FRET between Trp and Bim probes. Cartoons showing the possible degeneration of the ColE1c Trp fluorescence by a FRET mechanism in the presence of the bimane moiety. (A) Depiction of the average location of two stable conformers of the side-chain of a Trp residue, TrpA (red) and TrpB (blue), and a bimane probes Bim1 (dark gray) and Bim2 (light gray), all by rotation of the CaeCb dihedral. The lines represent the distance between two arbitrary atoms representing the electronic center of each probe. The vectors sketch the emission transition dipoles (green) and the absorption transition dipoles (yellow) in each probe, respectively. (B) Fluorescence lifetime distributions of the Trp conformers (donor alone, filled distributions), emitting with a bi-modal distribution lifetime, hypothesized as a long-lifetime and high-populated conformer (TrpA, red distribution centered at tA) and a short-lifetime and low-populated conformer (TrpB, blue distribution centered at tB). In the presence of bimane (donor-acceptor pair), the Trp lifetime distributions would correspond to four distributions (hollow distributions), each with probability given by the joined probabilities of the constituted conformers in the pair, and centered at the resulting lifetime tij (i ¼ A,B; j ¼ 1,2), according to the FRET efficiency. This efficiency is determined by the distance and relative orientation of the transition dipoles for the donor and acceptor conformers, as well as by the quantum yield of the donor conformer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

previously stated. The importance of the correct assignment of the origin of each component of the donor-acceptor signal will be relevant in the calculation of the FRET distances, as presented in the next section.

3.4. FRET distances for the soluble state of ColE1c The ‘global’ FRET section of the upper panel of Table 2, shows the spectroscopic parameters and average lifetimes needed to calculate the standard FRET distances in the soluble form of ColE1c. The €rster distances R0 (herein called the ‘global’ R0), and the transfer Fo efficiencies, were used for the calculation of the FRET distances by means of Eqs. (3e7). The ‘global’ FRET distances range from 24.5 Å in the Trp424/Bim361 pair, to 48.2 Å in the Trp460/Bim346 pair. Notably, all these distances are higher than the CbeCb distances obtained from the X-ray crystal structure (dbb, Table 2). The deviations are significant (±11.3 Å on average), even considering the molecular dimension of both probes and assuming that each pair face away from each other, maximizing the average separation between their electronic centers. Obviously, additional corrections and/or assumptions in the FRET calculations must be considered in

order to reconcile both sets of distances. In this sense, if it is considered that the two slower components of the Trp decay in the presence of the bimane acceptor (t2,DA and t3,DA) originate from the slowest decay component of the donoralone variant (t3,D); these three signals can then be used to calculate unbiased FRET efficiencies (Table 2, ‘t2/3 selective' FRET section). The ‘t2/3 selective’ efficiencies are higher than the ‘global’ ones, which imply distances closer to the corresponding R0 of the FRET pair. However, this latter parameter must also be calculated according to the quantum yield of the respective “selected” component of the donor. Thus, based on the fractional contribution of the slowest component of the donor-alone emission, f3, Q3 was calculated from QD, and likewise the corresponding R0 for each FRET pair. Altogether, the lower R0 values along with the higher efficiencies of the ‘t2/3 selective’ FRET calculations (Table 2), yielded significantly lower FRET distanceseaverage deviations of ±6.0 Å with respect to the X-ray CbeCb distances (dbb). However, a more realistic comparison between X-ray and FRET distances may involve taking into account the actual dimension of the probes and their “optimal” orientation in the protein. For this purpose, the same experimental bimane-labeled variants were

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Table 2 Spectral parameters and FRET distances between Trp donors and bimane acceptor of ColE1c by different FRET assumptions. FRET distances (dDA) from different FRET models between Trp (donor) and bimane (acceptor) for the soluble (upper panel) and for the membrane-bound (lower panel) forms. Spectral parameters for the “global” FRET were calculated according to eqs. (3)(7) for each case, and for the “t2/3 selective” FRET according to: aDA ¼ (a2$t2 þ a3$t3)/(a2 þ a3), bE ¼ 1(DA/D), c Q3 ¼ f3$QD, and dR0 calculated using eq. (5) with Q3 as donor quantum yield. In the case of the “t3 selective” FRET, the efficiency was calculated by gE ¼ 1(t3,DA/t3,D). For the soluble state, the reference distances (in Å) correspond to the eCbCb distance from the X-ray structure (PDB: 2i88), and to the fcenter-to-center distance of the modeled probes (see Fig. S3). For the membrane-bound state the reference was derived from the Ho model [33]. Soluble form at pH 7.0 Donor

Trp424

Trp460

Trp495

Acceptor

Bim347 Bim354 Bim361 Bim347 Bim354 Bim361 Bim347 Bim354 Bim361

“Global” FRET

“t2/3 selective” FRET

References

D

DA

E

QD

R0

dDA

t3,D

〈t2/3〉aDA

Eb

Qc3

Rd0

dDA

debb

dfcc

2.99 2.20 2.88 3.66 4.56 4.36 2.77 4.35 3.94

1.40 1.32 1.04 3.50 3.48 3.11 2.60 3.34 3.00

0.53 0.40 0.64 0.04 0.24 0.29 0.06 0.23 0.24

0.15 0.11 0.15 0.24 0.30 0.29 0.26 0.41 0.38

27.32 26.67 26.96 28.80 29.99 29.84 29.75 31.99 31.49

26.7 28.5 24.5 48.2 36.4 34.7 46.9 39.0 38.2

4.84 5.01 4.84 4.51 4.56 4.36 4.54 4.65 4.54

1.77 1.49 1.52 3.50 3.49 3.11 3.84 3.34 3.02

0.63 0.70 0.69 0.22 0.23 0.29 0.15 0.28 0.33

0.12 0.06 0.12 0.24 0.30 0.29 0.16 0.22 0.19

26.25 24.02 26.07 28.72 29.92 29.83 27.37 28.79 28.12

23.9 20.8 22.9 35.3 36.4 34.7 36.3 33.6 31.5

17.7 15.0 17.4 34.6 30.8 29.1 32.6 25.2 19.2

22.5 20.8 22.6 37.3 34.3 32.2 36.0 31.7 26.8

Membrane-bound form at pH 4.0 Donor

Trp424

Trp460

Trp495

Trp460 Trp460 Trp495

Acceptor

Bim347 Bim354 Bim361 Bim347 Bim354 Bim361 Bim347 Bim354 Bim361 Bim424 Bim495 Bim460

“Global” FRET

“t3 selective” FRET a 2/3〉DA

Reference g

D

DA

E

QD

R0

dDA

t3,D

〈t

E

Qc3

1.29 2.31 2.07 2.17 3.72 2.05 3.91 3.45 3.12 4.05 4.05 3.97

0.50 0.60 1.20 1.38 3.62 1.90 1.81 3.28 2.26 1.85 3.04 1.37

0.61 0.74 0.42 0.36 0.03 0.07 0.54 0.05 0.28 0.54 0.25 0.65

0.10 0.18 0.16 0.26 0.45 0.25 0.29 0.26 0.23 0.49 0.30 0.29

22.87 23.20 27.63 24.74 27.96 22.73 27.37 28.54 27.41 28.34 27.46 29.24

21.2 19.5 29.2 27.1 50.9 34.7 26.7 46.7 32.2 27.5 33.0 26.3

7.23 4.04 4.63 4.35 4.11 4.02 5.07 4.45 4.32 4.12 6.85 4.92

3.51 2.95 2.96 4.00 3.62 3.80 4.14 3.54 3.76 1.85 3.04 2.75

0.51 0.27 0.36 0.08 0.12 0.05 0.18 0.20 0.13 0.55 0.56 0.44

0.03 0.15 0.16 0.26 0.44 0.24 0.25 0.25 0.22 0.48 0.08 0.21

Rd0

dDA

dbb

18.63 22.43 27.50 24.72 27.89 22.63 26.71 28.44 27.19 28.24 22.07 27.61

18.4 26.5 30.2 37.1 38.9 36.4 34.3 35.7 37.3 27.3 21.3 28.7

30.07 38.82 47.94 11.38 9.00 16.33 18.09 19.86 25.52 40.4 18.1 36.1

Bold value indicates the experimental FRET distances according to the used assumptions.

produced in silico from the soluble X-ray crystal structure of ColE1c (PDB: 2i88). By using the Amber10-EHT force-field in MOE, the ideal geometry of the probe was obtained and these were energy minimized for the bimane moiety and the side-chains of neighbor residues of every FRET pair eall under an implicit solvent environment (Fig. S3). Table 2 includes the center-to-center distance, dcc, between the Trp indole (Cd-atom) and the bimane ring-system (N1-atom). The strong correlation between the distances reported by the structural model and by the ‘t2/3 selective’ FRET calculations is remarkable, with an average difference of 1.72 Å for the calculated distances compared with the modeled ones (Fig. 4A). This small difference is much lower than the dimension of the probes themselves, and can be accounted for by: (i) a mixed origin for the t2,DA signals; (ii) the arbitrary reference atoms used as probe electronic centers; (iii) differences between the soluble and the Xray structuresesoluble proteins are expected to be more expanded than crystallized ones; and (iv) deviation in the bimane conformer used in the model due to an incomplete sampling of the conformational space by the energy-minimization routine. Nevertheless, the calculated ‘t2/3 selective’ FRET distances harmonize the ones obtained by a computational model based on X-ray coordinates appear to validate the assumptions in the former calculations. On the contrary, the use of the standard method (‘global’ FRET) overestimated the inter-probe distances in this experimental system, and consequently would introduce significant errors in any mapping strategies derived from such distances.

3.5. Time-resolved measurements and FRET distances for the membrane-bound state of ColE1c The single-Trp variants of ColE1c showed a more homogeneous fluorescence decay in the membrane-bound forms than in the soluble forms (shaded rows in both panels of Table 1), which may correspond to a more similar environment around each emitter in the former than in the latter condition [32]. For the membranebound form, the bimane acceptor at the 354 site produces a larger effect on Trp fluorescence than at the two other positions (347 and 361) by reducing either the average lifetime of the Trp424 signal by the largest amount, and the average lifetimes of the Trp460 and Trp495 signals by the least amount. Thus, the ‘global’ FRET distances (Table 2, lower panel) reflected a pronounced ‘kink’ at position 354 for the Trp460/Bim354 and Trp495/Bim354 pairs, appearing as outliers in Fig. 4B (black line). This trend is curious since the 354 site is located in the middle of H1, where a mean distance with respect to the other two sites in H1 (347 and 361) would be expected regardless of the relative location/orientation between H1 and the Trp under consideration. The ‘t2/3 selective’ FRET distances, calculated according to the procedure defined for the soluble forms, yielded values with the same tendency (Fig. 4B, red line), although attenuated in comparison to the ‘global’ ones. In addition, most of the ‘t2/3 selective’ FRET distances in the membrane-bound form are shorter than the respective ones in the soluble protein (from the X-ray structure), which is unexpected since the protein expands upon membrane association [26]. Altogether, it is obvious that additional assumptions are required in the analysis of the lifetime decays, in order to get FRET distances for the membrane-bound form of ColE1c with meaningful values and

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Fig. 4. Comparison of distances in the ColE1c structure. X-ray, modeled and calculated FRET distances (circles) for the (A) soluble protein. The CbeCb distances correspond to the coordinates in the PDB: 2i88 structure, and the ‘center-to-center’ distances between the Trp and Bim centers of the modeled adducts based on the X-ray structure (see Fig. S3). (B) Membrane-bound protein. The ‘current’ model corresponds to distances derived from the model published by Ho et al. [33]. For further details, see the text and data reported in Table 2.

trends. In this sense, one possibility is that one of the bimane conformers might be underestimating the ‘t2/3 selective’ FRET distance, possibly by a geometric constraint that doesn't fulfill one/ some assumption(s) for the calculation, and might thus skew the FRET efficiency. For example, an orientation factor lower than the assumed isotropic average value (i.e., k2 ≪ 2/3, Eq. (5)), might be the case for one of the bimane conformers. The previous explanation is reasonable if it is considered that in the membrane-bound

form, the bimane probe might have a more restricted mobility due to interactions with the phospholipid headgroups. Another possibility is that the t2,DA decay has mixed contributions from the t2,D and t3,D decays, rather than only from t3,D, consequently decreasing the t2/3,DA average. It is also possible to consider that bimane shows just one stable conformer in the bound protein, from which the ‘t2/3 selective’ assumption would not be applicable. One or any combination of the previous hypotheses might bias the FRET

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distances. To overcome this problem, FRET calculations based only on the t3 components (t3,D and t3,DA), may eliminate, in principle, artifacts arising from mixed signals of different nature/origin. Indeed, the FRET distances calculated entirely from the t3 decays, ‘t3 selective’ FRET (Table 2, lower panel)ewhich correspond to the distances between the main conformers of each probee present “reasonable” values and tendencies (Fig. 4B, purple lines). However, when the ‘t3 selective’ FRET distances are contrasted with the CbeCb distances from the “current” model of the ColE1c closed-state, the Ho model [33], the average deviation is ~18 Å, ranging from 11.6 Å to 30 Å (Fig. 4B, orange line). Similar deviations are obtained with respect to the ‘global’ and ‘t2/3 selective’ sets of FRET distances. According to any FRET set herein reported (either ‘global’, ‘t2/3 selective’ or ‘t3 selective’), H1 should be further from Trp460 and from Trp495, but closer to Trp424, than appears in the current model. 3.6. An updated 3D-model of the membrane-bound form of ColE1c A new 3D-model of the membrane-bound form of ColE1c was needed in order to reconcile the set of FRET distances from which the current model was based on [26,33] and the new FRET distances obtained in this work. These sets encompassed FRET distances spread throughout the structure, and with different (i) molecular entities in the FRET pairs, (ii) pattern of labeling, (iii) experimental methodologies in the measurements (either by steady-state or time-resolved fluorescence), and (iv) assumptions in the analysis. Each set has its own strengths and weaknesses in terms of the ability to report accurate distances, which is related to the spectral properties and the size of the probes, probe interactions with neighbor residues, the effect of the Cyssubstitutions, and the effect of the labeling in the overall structure and dynamics of the toxin. The 9 FRET distances reported

herein were particularly relevant in the topology mapping given that (a) they involved naturally occurring probes (the Trp's), (b) Trp and bimane are small probes with relatively few rotatable bonds, and (c) all the probes, except Trp424, were located in stable secondary structure elements with known (calculated) lengths, tilt angles and depths in the membrane-bound form. All these factors constrained the model and limited the number of possible configurations. Nevertheless, all the data sets are potentially useful for a comprehensive mapping, and their simultaneous use afford the opportunity of an improved map in comparison with the employment of a subset(s) or independent use. Table 3 and Fig. 5 present the distances between the FRET pairs derived from the multilabeled new model of the closed-state of the channel-forming domain of ColE1, giving an average deviation of 1.19 Å with respect to the FRET distances. The new 3D-structure is depicted in Fig. 6, which fulfills the previous and new FRET distances in a 2Darrangement of external helices docked onto the membrane surface, in addition to the embedding geometry of helices and hairpins calculated for the Ho model [33]. The updated model reports a much lower distance between the DABMI426 (at H5) and Cou441 (at L5/6) pair than the FRET distance (3rd pair in Table 3). It's worthwhile to note that the actual experimental FRET pair corresponded to the opposite pair (i.e. Cou426/DABMI441). Nevertheless, this FRET pair was intentionally excluded during the modeling since (i) the need of alternating the coumarin and DABMI labels for a simultaneous fitting of all the FRET distances, and (ii) the FRET distance might be skewed by labeling at the 441 site with DABMI, rather than with the short sidechain of coumarin incorporated genetically into the protein. Thus, the conformation of the L5/6 loop might be “altered” due to the flexibility of the loop and the length of the probe. Indeed, the distortion that DABMI induces in the configuration of the L5/6 loop could be observed in the soluble form, where there is a difference of

Table 3 FRET and modeled distances for the membrane-bound form of ColE1c. Upper panel. Experimental FRET distances reported in the literature: the ‘inter-helices’ set by Ho et al. [33]a, the ‘509-radial’ set by Lindeberg et al. [26]b, and the ‘Trp-H1’ set from this work; and the center-to-centerc distance between the multi-adduct protein from the calculated model are reported as the new model (Figs. 5e6). The pairs #3 and #5 correspond to the inverted experimental FRET pairsd; and the pairs #5 and #10 were not included in the modelinge. Lower panel, for the validation, the experimental inter-Trp FRET distancesf were contrasted with the ones from the new and older models (Ho model [33]). #

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 #

1 2 3

Set

Interehelices

509radialb

TrpH1

Set

Inter-Trp

Structural elements

a

H1eH2 H2H3 H3eH4 d H4eH5 H5eL5/6 d,e L5/6eH6 L9/10eH1 L9/10eH3 L9/10eH4 L9/10eL4/5 e L9/10eL5/6 L4/5eH1 L4/5eH1 L4/5eH1 H6/7eH1 H6/7eH1 H6/7eH1 H9eH1 H9eH1 H9eH1 Structural elements

L4/5eH6/7 H6/7eH9 H9eL4/5

Bold value indicates FRET pairs not included in the modeling.

FRET pair

Distance (Å)

Don or Acc

Acc or don

FRET

Modelc

D

DABMI355 Cou369 DABMI390 Cou411 DABMI426 Cou441 AEDANS509 AEDANS509 AEDANS509 AEDANS509 AEDANS509 Trp424 Trp424 Trp424 Trp460 Trp460 Trp460 Trp495 Trp495 Trp495

Cou369 DABMI390 Cou411 DABMI426 Cou441 DABMI452 Trp356 Trp396 Trp413 Trp424 Trp460 Bim447 Bim354 Bim361 Bim347 Bim354 Bim361 Bim347 Bim354 Bim361

20.6 30.3 36.3 36.0 40.9 37.9 33.0 29.0 27.0 28.0 21.0 18.4 26.5 30.2 37.1 38.9 36.4 34.3 35.7 37.3

21.6 31.9 36.7 34.0 25.7 35.9 31.0 31.0 25.0 10.8 21.5 19.4 27.5 31.2 36.1 37.9 36.4 33.4 34.7 36.3

1.0 1.6 0.4 2.0 15.2 2.0 2.0 2.0 2.0 17.2 0.5 1.0 1.0 1.0 1.0 1.0 0.1 0.9 1.0 1.0

FRET pair

Distance (Å)

Don or Acc

Acc or don

FRET

New

Oldf

Trp424 Bim460 Trp495

Bim460 Trp495 Bim424

27.3 21.3 28.7

23.6 24.7 29.5

40.4 18.1 36.1

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Fig. 5. Inter-probe distances in the new 3D-model of the membrane-bound ColE1c. Ribbon representation of the new model of the membrane-bound ColE1c depicting 23 interprobe distances shown in Table 3 by connecting lines in the multi-labeled protein, colored as follows: bimane in orange, AEDANS in black, DABMI and coumarin in cyan, and Trp in mangenta, except for Trp424 in blue, Trp460 in red, and Trp495 in green. The red dots are a coarse-grained representation of lipid carbonyl groups of the outer leaflet of the plasma membrane. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

~20 Å between the FRET and the CaeCa X-ray distances [33], which cannot be accounted for by the actual dimensions of the probe. The new 3D arrangement of ColE1c on the membrane is in agreement with the H1eH5 ‘inter-helices’ set of FRET distances. To simultaneously fulfill the FRET constraints as far as possible, the experimental DABMI390/Cou441 FRET pair (5th pair in Table 3) was also inverted to the Cou390/DABMI411 in our in silico labeling scheme. In this case, though, the inverted pair might be reporting a similar distance to the original one, since both probes are located at a-helix structures rather than at flexible loops. Furthermore, the updated model reconciles quite well with the distances between the 509 site labeled with AEDANS (at L9/10) and 4 Trp residues at different locations. A first modeling attempt was performed by including the five AEDANS/Trp FRET pairs in Table 3; however, the pair that includes Trp424 (at L4/5) reported a significant restraint energy under different topologies and configurations of the L4/5 loopethe length of the AEDANS molecule and the closer proximity of L4/5 cannot fit the 28 Å separation reported by FRET, without affecting the actual distances between the others FRET pairs.

3.7. Validation of the new model of the membrane-bound form of ColE1c The FRET assumptions and calculations wereein principleevalidated when the FRET distances were compared with the ones obtained from the X-ray structure of the soluble form (Table 2, upper panel). Now, to validate a particular model, and even to compare closed-state models, it is important to compare an independent set of FRET distances (i.e., distances not used in the modeling) with the ones derived from the model(s). In this sense, the best FRET pairs for this assessment arise from permuting the three Trp residues used in the Trp/Bim pairs, since they are common nodes on the network of inter-residues vectors of the ‘TrpeH1’ set. The lifetime measurements of Trp460/Bim424, Trp460/Bim495 and Trp495/Bim424 FRET pairs in the membrane-bound form (Table 1, bottom panel), were used to estimate inter-Trp distances (Table 2, bottom panel). As observed, neither the ‘global’ nor the ‘t3 selective’ sets of FRET distances match the inter-Trp distances in the old model (Table 3, lower panel), given the 7.9 Å of average deviation. In contrast, when the ‘t3 selective’ inter-Trp FRET distances are compared with the ones in the new model, the deviations are 3-

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which is thought to arise as a slow 2D-arrangement of the surface helices, was deliberately omitted since it corresponds to a postinsertion step, and its description does not contribute to the integration phenomenon itself. In this section, simulated transient signals are shown that reproduce diverse observed phenomena of the integration of the soluble state (E1) to the inserted membranebound state (E6), and then the ability of the proposed kinetic scheme was used to explain the sets of recorded signals. Firstly, after rapid mixing of ColE1c with LUVs constituted with bromide di-substituted lipid acyl chains [34], the observable “membrane-quenching” transition, Mq, was simulated with Gaussian noise by

    Mq ðtÞ ¼ Qunq $ Eunq þ Qqnc $ Eqnc þ Gð0; sÞ

(13)

with unq corresponding to the unquenched state, and qnc to the quenched state. [Ei] corresponds to the fractional concentration of ColE1c, and Qi to the relative molar fluorescence for the state “i”. The function G(0,s) is a noise generator with Gaussian distributed random values with media zero (0) and variance s. For this particular observable, Qqnc was set to 0 since there is no fluorescent signal at a longer incubation time [34], and with the conversion unq / qnc following a unidirectional mono-exponential relaxation defined by kobs ¼6:6s1

Eunq !Eqnc

with

  Eunq  ¼ 1 Eunq ¼ 0

at at

t¼0 t¼∞

 (14)

Then, the synthetic observable was fit to a multi-state function defined by Fig. 6. New 3D-model of the membrane-bound ColE1c. Ribbon representation of the new model of the membrane-bound ColE1c based on 18 FRET distances in Table 3, and the “vertical” position of the helices and hairpins in Table S1. H1eH7 and H10 lay on the membrane surface, while the H8/9 hairpin orients perpendicular to the membrane surface and is transmembrane. (A) Perspective view. The gray dots represent the plane of the outer membrane layer. (B) Upper view from the extracellular side. In both, the ribbons are spectrally colored from blue (N-terminus) to red (C-terminus). The green vectors depict the inertial axes of H6eH9. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

fold lower, with an average value of 2.6 Å. This value is higher than the average deviation of 1.19 Å obtained from the “modeling set”. However, they are reasonably similar considering that the experimental distances come from the Trp/Bim FRET-pairs, while the modeled distance come from the Trp/Trp pairs. The bimane adducts have the additional CeSeC atoms as part of their side-chains (with an average CeC distance of ~2.8 Å) with respect to the Trp residues. 3.8. Kinetic validation of the integration mechanism A sequential mechanism has been proposed for the in vitro integration of the pore-forming ColE1c in model membranes [45] by aggregation

binding

unfolding

elongation

insertion

condensation

E1 ! E2 ! E3 ! E4 ! E5 !E6 ! E7 (12) The kinetics of the transition of the soluble insertion-competent form (at pH 4.0 and 0.1 M ionic strength) to the membrane-bound form in LUVs with 1000 Å of average diameter, were measured by monitoring the fluorescence signals after stopped-flow mixing in various experimental designs [26]. Table S2 summarizes the rate constants and the associated events according to the criteria of the respective authors, and Fig. 7 shows the transitory fractional concentration of each intermediate. The condensation step, E6 / E7,

FðtÞ ¼

6 X i¼1

 Qi $½Ei ðtÞ

with

½E1  ¼ 1 ½E1  ¼ 0

at at

t¼0 t¼∞

 (15)

being [Ei] the fractional concentration of ColE1c for the intermediate “i”, with temporal changes dictated by the sequential reactions in Eq. (12), and the first-order rate constants defined in Table S2. In Eq. (15), the relative molar fluorescence of each intermediate, 0  Qi  1, is inversely related to the effective collision probability between the Trp residues and any of the Br-atoms of the lipid acyl chains. Fig. 8 shows the fit (black solid lines) of different models of “membrane-quenching” (M1eM12, Table 4), to the unique synthetic fluorescent transient Mq(t) (colored noisy curves). In the models M1 and M8, the quenching occurs in an all-or-none fashion. In M1, the quenching is delayed until the formation of the E6 intermediate (Q1..5 ¼ 1 and Q6 ¼ 0). Despite the fact that this model is physically-appealing, since E6 is the “expected” state to interact with the embedded quencher into the bilayer, the evident deviation between the ‘observed’ and the fitted curves rules out this mechanism. In other words, the so-called inserted state proposed by Lindeberg [26], cannot account for all the quenching caused by the labeled lipids. On the contrary, in M8, the onset of quenching appears as early as the first insoluble intermediate, the E2 state (Q1 ¼ 1 and Q2..6 ¼ 0). Obviously, this model lacks physical meaning since it assigns the so-called aggregated stateethought as an aggregation of the soluble protein onto the membrane surface [34], a quenching efficiency as high as the inserted state. The other models are, in principle, reasonable since they consider the membrane quencher to be totally ineffective against soluble-like states (Q1..2 ¼ 1, e.g. M2eM6 models), and/or totally effective against inserted-like states (Q4..6 ¼ 0 or Q5..6 ¼ 0, e.g. M5eM7 models). However, the fit of the free-floating parameters (Q2eQ5, according to the case) failed to describe the observable mono-exponential trend, as follow: (i) models M2, M4 and M5 show biphasic quenching; (ii)

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Fig. 7. Time-course of the intermediates during the integration of ColE1c. Simulated transients in the fractional concentration of the 6 states (E1eE6) during the sequential integration process from the fully soluble state (E1 ¼ 1 at t ¼ 0) to the fully inserted state (E6 ¼ 1 at t / ∞). Reaction scheme (Eq. (12)) and rate constants (in s1, Table S2) shown in the upper panel.

models M6 and M7 deviate around the characteristic time (1/kobs); (iii) model M3 seems to adequately fit the model, although with a clear trend of the residual distribution; and (iv) models M10 and M11 also fail to statistically describe the residuals (Table 4, not plotted). Only models M9 and M12 show a statistically acceptable fit, with M9 reflecting the best performance. According to model M9, the ‘membrane-quenching” comprises the differential participation of all the intermediates, and occurs even in the first E1 / E2 step (3.7% of fractional change), and with the major change occurring during the E3 / E4 transition (39% of fractional quenching). Paradoxically the last step, E5 / E6, represents less

kobs ¼180s1

kobs ¼30s1

Eunq !Eint !Eqnc

 with

    Sq ðtÞ ¼ Qunq $ Eunq þ Qint $½Eint  þ Qqnc $ Eqnc þ Gð0; sÞ

with unq corresponding to the unquenched state, int to the intermediary state, and qnc to the quenched state. [Ei] corresponds to the fractional concentration of ColE1c, and Qi to the relative molar fluorescence for the state “i”. For this particular observable, Qqnc was set to 0.06 since there is a residual fluorescent signal at a longer incubation time [34], and the conversion follows a unidirectional bi-exponential relaxation defined by

   ¼1  Eunq Eunq ¼ 0:06

than 10% of the fractional change. Equivalently, the quenching of the Trp fluorescence of ColE1c by the TNP moiety bound to the lipid headgroups, constituting “surface-quenching”, Sq, was simulated with Gaussian noise by

(16)

at at

t¼0 t¼∞

 (17)

Then, the synthetic observable, Sq(t), was fit to the multi-state function defined previously (Eq. (15)) with the relative molar fluorescence of each intermediate, 0  Q  1, inversely related to the FRET efficiency between the Trp residues and the TNP moieties on the membrane surface. Fig. 9 and Table 5 show different models

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Fig. 8. Trp “membrane-quenching” during the integration of ColE1c. Synthetic transients (noisy curves) of the Trp fluorescence of ColE1c after rapid mixing with LUVs with Brlabeled lipids by using the observed rate of kobs¼ 6.6 s1 (taken from Zakharov et al. [34]), and Eqs. (13e14), The fitting of the simulated transient to 9 quenching models (M1eM9) by using a 6-state sequential model to the observable defined by Eq. (15) is represented by a solid curve in each case. In each quenching model, the relative molar fluorescence of each state, Qi, is either fixed (red parameter(s)) or free-floating (blue parameter(s)). Reaction scheme (Eq. (12)) and rate constants (in s1) shown in the upper panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 4 Fit of the “membrane-quenching” transient to different models of quenching. Upper panel: Relative molar fluorescence for each intermediate, Qi, of the sequential mechanism in eq. (12), according to the fit of the synthetic transient to eq. (15) (Fig. 8), of 12 quench models, M1M12, by different assignment (fixed parameters) of the unquenched states (light shaded cells) and the quenched states (dark shaded cells). The clear cells correspond to free-floating parameters. c2 corresponds to the reduced chisquare, and p to the p-values statistics. Lower panel: fractional fluorescence change, (QiQj)100, for the Ei/Ej step.

of “surface-quenching”. Models S2 and S4 are statistically satisfactory, with model S4 being slightly better. This model is physically appealing since it reports a ~70% fractional change in the quenching efficiency to the first E1 / E2 transition, ~30% to the next E2 / E3 step, and a marginal change to the E3 / E4 transition. Further steps occurs without changes in the quenching efficiency. Furthermore, the proposed kinetic scheme for the integration was assessed by its ability to describe a set of transitory FRET efficiencies between single-Trp (donors) at different positions and AEDANS adducts at Cys509 (acceptor) after rapid mixing of ColE1c

with LUVs [34]. For this, the primary observable corresponded to a continual change of the donor fluorescence in the presence of the acceptor, FDA, according to the inter-probe distance, R(t), by

2

3

6 FDA ðtÞ ¼ FD 41 

1 1þ

RðtÞ R0

7

6 5

(18)

with FD representing the fluorescence of the single-Trp ColE1c in €rster distance for the the absence of the acceptor, and R0 the Fo

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Fig. 9. Trp “surface-quenching” during the membrane integration of ColE1c. Synthetic transients (noisy curves) of the Trp fluorescence of ColE1c after rapid mixing with LUVs with TNP-labeled lipids, by using the observed rates of kobs,1 ¼ 180 s1 and kobs,2 ¼ 30 s1 (taken from Zakharov et al. [34]) and Eqs. (16e17). The fitting of the simulated transient to 4 quenching models (solid curves, S1eS4) by using a 6-state sequential aggregation model to the observable defined by Eq. (15) is represented by a solid curve in each case. In each quench model, the relative molar fluorescence of each state, Qi, is either fixed (red parameter(s)) or free-floating (blue parameter(s)). Reaction scheme (Eq. (12)) and rate constants (in s1) are shown in the upper panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 5 Fit of the “surface-quenching” transient to different quench models. Upper panel: Relative molar fluorescence for each intermediate, Qi, of the sequential mechanism in eq. (12), according to the fit of the synthetic transient to eq. (15) (Fig. 9), of 5 quench models, M1M12, by different assignment (fixed parameters) of the unquenched states (light shaded cells) and the quenched states (dark shaded cells). The clear cells correspond to free-floating parameters. c2 corresponds to the reduced chi-square, and p to the pvalues statistics. Lower panel: fractional fluorescence change, (QiQj)100, for the Ei/Ej step.

Table 6 Fit of 509AEDANSeTrp FRET distance transients to a 6-state sequential model. Upper panel: Nominal distance (in Å) for each intermediate of the sequential mechanism in eq. (12), according to the fit of the synthetic transient of five 509AEDANSeTrp distance relaxation (Fig. 10) to eq. (20). The light shaded cells correspond to the initial distance, and the dark shaded cell to the final distance, from Lindeberg et al. [26]. The clear cells correspond to free-floating parameters. c2 corresponds to the reduced chi-square, and p to the p-values statistics. Lower panel: fractional distance change, (DiDj)100, for the Ei/Ej step.

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Fig. 10. Inter-probe distances during the integration of ColE1c. Synthetic transients (noisy curves) of the AEDANS509/Trp FRET distances, after rapid mixing of single-Trp ColE1c variants with LUVs by using the observed rates: kW356 ¼ 25.2 s1, kW396 ¼ 13.1 s1, kW413 ¼ 12.0 s1, kW424 ¼ 6.1 s1, kW460 ¼ 12.5 s1, and kW495 ¼ 38.9 s1 (taken from Lindeberg et al. [26]) for each FRET pair, and Eqs. (18e19). The fit of the simulated transient by using a 6-state sequential model (Eq. (12)) to the observable defined by Eq. (20) is represented by a solid curve in each case. In each quench model, the nominal inter-probe distance of each state, Di, was either fixed (red parameter(s)) or free-floating (blue parameter(s)). Reaction scheme (Eq. (12)) and rate constants (in s1, Table S2) are shown in the upper panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

particular FRET pair. The inter-probe distances were simulated to relax mono-exponentially from Ro (in the soluble form, E1) to R∞ (in the integrated form, E6) according to

RDA ðtÞ ¼

ðkobs $tÞ

RðtÞ ¼ R∞ þ ðRo  R∞ Þ$e þ Gð0; sÞ

with



R ¼ Ro R ¼ R∞

the distance associated with the sequence of events that describe the integration process (Eq. (12)) by

at at

t¼0 t¼∞



6 X i¼1

(19)

The continual changes in the inter-probe distance between Ro and R∞ for a particular FRET pair was fitted by discrete changes in

 Di $½Ei ðtÞ

with

½E1  ¼ 1 ½E1  ¼ 0

at at

t¼0 t¼∞

 (20)

In this case, [Ei] corresponds to the fractional concentration or fractional residence time of the intermediate “i”, and Di its nominal inter-probe distance, with Ro  Di  R∞ for expansions, with Ro  Di  R∞ for contractions. Table 6 shows the nominal inter-

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probe distance of each FRET pair that best fit each transition (Fig. 10). Most transitions can be described by 2 different intermediates with respect to the inter-probe distance; the exceptions are Trp495 (1 intermediate) and Trp424 (4 intermediates). For all the FRET pairs, the onset of changes in the inter-probe distance appears in the first step (E1 / E2), being significant for Trp495 with a fractional change of ~36%. However, the distances for the FRET pairs with Trp356 and Trp495 have their major change in the E2 / E3 transition, while for Trp396, Trp413 and Trp424, the major changes occur in the E3 / E4 step. Notably, for all the FRET pairs, except with Trp424, the complete change in the distance occurs during the formation of the first four intermediates, E1 / E4. 4. Discussion In this study, we propose a new all-atom model of the membrane-bound close-state of the ColE1 channel-forming domain, which reviews the general properties described in previous models and is summarized as the topology denominated “umbrella model”. The new 3D-structure is depicted in Fig. 6, and is outlined as: (i) A membrane anchor core formed by H8 and H9 immersed into the membrane bilayer with embedding geometry preserved from the Ho model [33]. Thus, these two a-helices will be used as a common reference framework for the purpose of comparison between the models. (ii) A circular-like arrangement of the peripheral a-helices around the H8/9 central hairpin, which resembles the original 2D-topology proposed earlier [26] and later updated by our group [33], i.e., the “umbrella-like” model. However, a remarkable similarity with the former model, yet different from the latter model, is the counter-clockwise direction (from an extracellular view) of H1eH7 around the central H8/9 hairpin (see Fig. S4). (iii) The same embedding geometry of the external a-helices, i.e., tilt angles and depths calculated in the Ho model. (iv) The configuration and vertical position of the hairpins H6/7 and H8/9 are preserved from the Ho model. However, in contrast, the new model preserves also the H1/2 hairpin super-structure from the soluble form. (v) The new 3D-structure is more compact than previous models, allowing contact among most of its secondary structure elements with clusters of a-helices around the central H8/9 anchor, instead of around H1 [33]. In our earlier proposal of the membrane-bound ColE1c, H1 and H2 were dissociated on the membrane due to their higher (more negative) transfer energy than as a hairpin entity [33]. However, the transfer DG of the H1/2 hairpin to the membrane is still negative, and it must be taken into consideration that the transfer calculations were made for isolated entities (either a-helices or a-hairpins), out of the context from the rest of the protein. In particular, the “compactness” of the new topology allows interaction of H1 with H8/9 that might serve to stabilize the hairpin configuration of H1 and H2. The same FRET distance between 355DABMI (at H1) and 369COU (at H2) in both the soluble and the membrane-bound proteins [33], and the same pattern of mobility of the segment 355e363 (the tip H1/2 hairpin) between the soluble and the membrane-bound states [18,19], support the preservation of the H1/2 super-secondary structure. Besides, the location of the H1/2 hairpin the new model is also compatible with a comprehensive mechanism of insertion and unfolding on the membrane (see below). In addition, the modeling employed an efficient conformational search protocol to assess the conformational flexibility of the sidechains of residues and probes, in addition to the main-chain of random loops. The new model depicted in Fig. 6 represents the conformation with the lowest internal energy found by the search methodology (i.e., the obtained “global minimum”). Thus, an ensemble of tens of slightly different conformations (i.e, obtained

69

“local minima”) are available, which conformers do not differ from each other by more than 0.005 kcal/mol of internal energy per heavy-atom, and with an absolute difference in the restraint energy lower than 0.2 kcal/mol. 4.1. Functional relevance of the new features of closed-state model in the context of the open-state channel The updated model of the closed-state ColE1 C-domain shows a 2D-array on the membrane that points toward an analog function of H1/2 and H6/7 in the open state channel, since their clustered configuration aroundebut antisymmetric with respect toethe central H8/9 membrane anchor. In this sense, photolabelling [49]; biotinylation [50]; [51]; epitope mapping [52]; saturation mutagenesis [53] protease accessibility [54]; depth-dependent fluorescence quenching [25,55] cysteine-scanning mutagenesis [28]; FRET [24]; CD, Fourier transform infrared radiation, and differential scanning calorimetry [6,11]; time-resolved spin labeling [7]; and acrylamide quenching and other fluorescence data [22] help to explain the transition to the open-state channel as a voltageinduced insertion of part or all of the H1eH2 and H6eH7 segments to form the TM1 and TM2 transmembrane “helices”, respectively. These two TM-segments are proposed to stabilize the open channel along the voltage-independent insertion of H8 (TM3) and H9 (TM4) helices (or H8/9 helical hairpin). This large conformational scale change has also been reported for colicin Ia [50], and there is a consensus that the colicin channel consists of 6 out of 10 original (soluble) helices [27]. From all the previous results, the location of H1eH2 (or the H1/2 hairpin) is relevant to its putative role in forming part of the channel pore. A report on colicin Ia identified 11 key channel-lining residues in the hydrophobic segment that affects the channel conductance and/or selectivity [56]. Notably, after sequence alignment of the Ia and E1 colicins, the corresponding residues in the H8/ 9 segment of ColE1c face the H1/2 hairpin in the new model. From the premise that the ColE1c monomer is the functional unit, this evidence supports the new closed-state model in terms of the participation of the H1/2 hairpin in directing the TM1 interaction with the TM3eTM4 elements. 4.2. New insights in the mechanism of integration The soluble protein possesses, at a pH value lower than 4.5, an enhanced capability to approach and bind to anionic membranes. Zakharov [34] and Lindeberg [26] recorded an invaluable set of FRET transitions from various probes and experimental designs, which enabled the monitoring of different aspects for the integration (soluble / inserted) process. These researchers proposed a set of sequential and uncoupled integration events, picturing the unfolded protein as an extended array of the a-helices on the membrane surface that precede the insertion of the hydrophobic hairpin [8,34,57]. In this section, we interpret the kinetic evidence in light of the updated closed-state model, to elicit a new integration mechanism of the ColE1c in synthetic membranes under acidic activation. Given the 7-state mechanism pertaining to Eq. (12) and the experimental rate constants in Table S2, the best model that describes the phenomenon of “surface-quenching”, model S4, reported three transitions with changes in the efficiency of the quenching (see Fig. 9). Zakharov [34] dismissed a mechanistic relevance for the fastest observed transition (kobs ¼ 180 s1), attributing it to a FRET quenching of the Trp signal associated with the high R0 for the FRET pair, and to the “accumulation of Colicin E1 molecules in the near-membrane space”. However, the fit reported an unequivocal high contribution of fractional quenching (~69%) to the

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Fig. 11. (A) Layers constituting the ColE1c soluble structure. Views of the X-ray ColE1c structure depicting layerA (H1eH2 and H10 helices, blue ribbons), layerB (H5 and H8eH9 helices, red ribbons), and layerC (H3, H4 and H6eH7 helices, cyan ribbons). (B) Model of ColE1c approach to the membrane surface. Suggested orientation of the soluble ColE1c during its approach and docking to the membrane plane (green surface). The figure shows four acidic residues, among others, that face the bottom part of the protein. It is notable the angular orientation of H1, and the parallel orientation of the inertial axis of H4 to the membrane plane. Some helices are not depicted. (C) Model of the H6/7 hairpin opening at the membrane surface. Hypothetical trajectory of the H6/7 hairpin by a relative lifting (ribbons colored spectrally from blue to red) of the helical structure while H8/9 (red ribbons) is integrated into the membrane bilayer. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

E1 / E2 transition. This quenching corresponds to the approach of Trp emitters of ColE1c to the labeled polar group with an effective distance of ca. 0.87R0 (~23 Å, given the R0 ¼ 27 Å for that FRET pair). Considering the width of the interfacial polar regions of the membrane (~15 Å), and the excluded-volume of the protein, we propose that this early event corresponds to a closer contact (herein called the absorption step) of the protein to the membrane that produces structural changes in the former, as observed also in the inter-probe distance transient (see Fig. 10). Elkins [8] suggested the in vivo role of the H1eH2 segment as a “structural hinge” around which the channel domain could rotate into position to contact the membrane surface by means of layerC (helices H3eH4 and H6eH7). However, the angular orientation of H1 in relation to the H8/9 axis (see Fig. 11B) is compatible instead with the role of a “structural arm” that allows the perpendicular orientation of the central hydrophobic helices on the membrane surface. Thus, we endorse the idea that the soluble insertion-competent ColE1c approaches the membrane by positioning its bottom surface in close proximity to the membrane surface, where the central H8/9 hairpin would initiate the spontaneous insertion into the bilayer, which has been originally formulated for colicin A [10]. This

physical approach would be driven by the electrostatic complementarity between both surfaces, due to the overall positively charged protein surface and the negatively charged membrane surface [57]. Herein, we propose that candidate residues for a pHtrigger role that can respond to acidification might be all or part of the Glu cluster (Glu361 at H1, Glu365 at H2, and Glu518 at H10), along with Asp405 and Asp410, that faces the bottom surface of the soluble protein (see Fig. 11B), and is in close proximity to the apex of the H8/9 hairpin. Indeed, the charged (deprotonated) Glu361 and Glu518 at pH 7.0, are neutralized by an in silico protonation at pH 4.0 [44]. Remarkably, Glu361 is conserved in the E- and A-type colicins, while Glu365 and Glu518 appear in some E-type toxins. A perpendicular orientation of the soluble ColE1c relative to the membrane plane would allow a progressive anchoring of the hydrophobic H8/9 hairpin into the hydrophobic core of the bilayer. The ~4% of fractional “membrane-quenching” during the docking transition, E1 / E2, also suggests the direct interaction (collisional contact) between the Trp(s) of the insertion-competent ColE1c and the Br-atom(s) of the membrane lipids. The Br-atoms at C9 and C10 in the acyl-chains of the phospholipids are ca. 8 Å from the center of the bilayer with a range of thermal motions of ca.

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±5 Å [34]. Accordingly, they are able to marginally collide (i.e., with low frequency/efficiency) with a Trp probe during the early stage of the integration as long as this putative residue is located at the contact point of the protein on the membrane surface. Due to its location close to the apex of the H8/9 hairpin, and the putative approach mechanism, Trp495 would be the residue (among the three naturally occurring Trps) that would first contact the membrane during this early stage. In support of this mechanism, the change in the distance between Trp495 and AEDANS509 (at L9/10) monitored by FRET transients, presents the highest kobs among six probes located throughout the protein (see Fig. 10). Although, it is important to stress that all the transitory TrpeAEDANS509 distances reflected changes during the E1 / E2 transition (see Table 6), which is remarkable since this shows that the earliest contact with the membrane is able to elicit a slight loosening of the external layers with respect to the central element (layerB). Nevertheless, the case of Trp495 is relevant and requires special attention, since it presents the highest fractional change in the distance to AEDANS509 among all the six probes tested. This might have its explanation in a slight side-chain re-configuration of any member(s) of the FRET pair, associated with the initial contact of the bottom surface of the protein with the membrane surface, and which might correspond to the Trp495 transit time through the variable-dielectric interfacial layer. The rest of the fractional change in the Trp495eAEDANS509 distance occurs during the subsequent E2 / E3 transition, which we propose as the onset of the insertion process itself (discussed next). The following transitions, E3 / E6, elapse with steady kinetics, which is compatible with the progressive perpendicular embedding of the H8/9 hairpin at an invariable Trp495eAEDANS509 distance (ca. H9 length) into the constant, dielectric membrane bilayer core. Obviously, for this reason, this particular signal is not a good reporter of the whole insertion process. Moreover, the bottom region of the soluble form is a relatively “flat” surface, where the frontal and parallel H4 would contact the membrane (see Fig. 11B), and the electrostatic repulsion of its two Asp residues (Asp408 and Asp410) with the negatively charged membrane might act as lever to force/induce the opening of the entire H3eH5 segment. The variants, D408S and D410S, indeed showed an impaired ability to bind the membrane in support of this idea [37]. At pH 4.5, the D408S variant is impaired 10-fold in membrane binding, while the D410S is impaired 3-fold compared with the WT protein [37]. These observations are compatible with the greater solvent exposure of the Asp408 residue. If Trp495 is the main reporter of the “membrane quenching” during the initial stages, then, according to the M9 model, the fractional quenching during the E2 / E3 transition (~21%) would correspond to the progress of the H8/9 insertion. This process necessarily implies the disruption of their hydrophobic interactions with the external and amphipathic H1eH7 helices, which gradually substitute their inter-helical contacts with interactions towards membrane elements (phospholipids, hopanoids, etc.). This insertion event would expose the hydrophobic inner face of the amphipathic helices to the hydrophobic bilayer region, while locating the hydrophilic residues to the interfacial zone, displacing and rearranging the distribution of phospholipids around the protein. All this pertains to the second and the slowest transition observed by the “surface-quenching” (kobs ¼ 30 s1), where the remaining fluorescence from the Trp424 and Trp460 residues (~30%) are quenched by the TNP probes. We hypothesize that the opening of the outer helical layers, layerA (H1eH2 and H10) and layerC (H3eH4 and H6/7), that surround the central layerB (H5 and H8/9) (Fig. 11A, upper view), occur by

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pivoting the complete H3eH7 segment with a hinge located at the short L7/8 turn, while preserving the inter-motif H6/7 and H8/9 configuration as observed in the crystal structure (Fig. 11C) [33]. However, in the folded soluble ColE1c structure, the compact H6/7 hairpin is embedded concentrically into the outer H3eH5 segment (Fig. 11A, right side view), which would first contact the membrane surface during the anchoring of H8/9. Incidentally, the GNM and ANM models predict a high mobility for the H3eH5 segment in the soluble form, despite the observed fluctuations in the X-ray structure (B-factors) that are attenuated by crystal contacts (Fig. S5). We might speculate that the upward movement of this H3eH5 segment is hinged at the L2/3 loop and L7/8 turn, and may be concerted along the entire H3eH7 segment, or in phases by raising the H3eH5 outer segment first, followed by the inner H6/7 hairpin (Fig. 11C). In the soluble ColE1c, His440 is located in the L5/6 loop and by H-bonding with Asp473 at the C-terminus of H7 forms a “neck’ that closes the H6/7 hairpin. It has been suggested that the protonation of some of the neutral/acid residues might eliminate key salt-bridges needed for the stabilization of the soluble structure e becoming an insertion-competent state in the soluble channel domain. The protonation of a His residues at pH below 6.0 has been reported [58], so it is feasible that protonation His440 in acidic mediaealong with the protonation of Asp473emay induce dissociation of the L5/6 loop from H7, and consequently the longer L5/6 loop could act as a hinge that would allow the independent elevation of both inner (H6/7) and outer (H3eH5) segments. On the other hand, the H1eH2 segment (or the H1/2 hairpin) and the terminal H10, which forms the layerA (Fig. 11A, left side view), may spontaneously unfold with hinges at the L2/3 and L9/10 loops. For colicin A, an independent opening of H1eH2 in an early step of the insertion process has been reported [27]. Since the original work of Elkins [8], the outer layers of the soluble structure have been recognized and characterized as units for unfolding of ColE1c onto the membrane surface. Moreover, Elkins stated that layerC interacts firstly with the membrane surface, in agreement with our hypothesis. However, Elkins [8], Zakharov [34] and Lindeberg [26] considered that the E2 / E5 transitions correspond to the unfolding of protein into an extended array of a-helices on the membrane surface that precede the insertion (E5 / E6 step) of the hydrophobic hairpinethe sequential unfolding-insertion mechanism. In summary (i) model M9 revealed that each intermediate is differentially affected by the membrane-quencher, which allows a complete monitoring of the integration process; whereas, as suggested by Lindeberg [26], the E5 / E6 step is the transition associated with the formation/ appearance of the speciesethe inserted state (see Fig. 7), susceptible to the embedded quenchers. Correspondingly, in colicin A, the three Trps are on the H3eH7 segment, and the “membranequenching” is observed even when the insertion of the H8/9 hairpin is precluded [28]; (ii) the invariability of the Trp495eAEDANS509 distance during the E3 / E6 steps, which is understood as the displacement of the H8/9 hairpin throughout the constant dielectric of the membrane core (the embedding), while the variable distances during the earlier two steps (E2 / E4) for the rest of the probes, reflect a re-arrangement of the protein tertiary structure (the unfolding); and (iii) the invariability of the “surfacequenching” during the last two transitions, E4 / E6, which can be understood as the formation of the closed structure on the membrane that occurs in two earlier steps. We propose an alternative mechanismethe concerted unfolding-insertion integration mechanism, mainly occurring during the E2 / E3 / E4 transitions. These two transitions represent 65%ee100% of the remaining fractional change of each signal. Altogether, after the

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absorption of the soluble protein onto the membrane surface during the E1 / E2 transition, there is a concurrent unfolding of the external layers and insertion of the hydrophobic elements of the protein during the E2 / E3 / E4 transitions, which is congruent with the absence of unfolding of the peripheral helices when the membrane fluidity is sufficiently low [26]. This observation supports the idea that by suppressing lateral phospholipid displacements, the immersion of the hydrophobic H8/9 hairpin is prevented, and concomitantly the unfolding of the protein (i.e., the unfolding does not precede the integration as stated in the sequential mechanism). The detection of the E4 / E5 / E6 transitions by the ‘membrane-quenching” phenomenon, might correspond to an advanced state of re-arrangement of the unfolded protein, and must arise from the Trp424 and Trp460 signals. However, only the changes in the Trp424eAEDANS509 distance correlate with alterations in the “membrane-quenching” for these transitions. Thus, the E4 / E5 transition (~26% of fractional quenching) might have its origin to the transition of the L4/5 loop (the attachment point of Trp424) to a helical structure upon membrane binding, the elongation step [7], which would favor the interaction of Trp424 with membrane quenchers. The E5 / E6 transition corresponds to a further minor adjustment (only ~9% of the fractional quenching) of the already unfolded and integrated protein. Separately, it has been reported in a longer kinetic window observation that there is a slight decrease in the distance of the H3eH5 segment to the central coreethe condensation step, E6 / E7, with a kobs of 0.5 s1 [26]. Our kinetics analysis doesn't consider explicitly this step without detriment of the rest of the time courses, since it is uncoupled to the predecessor step, i.e., k6/7/ k5/6 < 0.1. Nevertheless, our model is compatible with this slow rearrangement of the H3eH5 segment, while preserving the stability of the other segments. Indeed, our proposal states that the intrinsically stable H1/2 and H6/7 super-secondary structures are in close proximity to the central H8/9 core from the onset of the integration (E2 / E3), and there is no need for further re-packing of these elements. Remarkably, the old model presents a topology where residues belonging to the H1eH5 segment are facing in the opposite direction in comparison with the soluble form. Such a large rearrangement would require a counter clockwise spiral within the protein in order to unwind the H1eH5 segment (Fig. S6). 5. Conclusions Our updated model of ColE1c based on the “umbrella” configuration of the two-dimensional arrangement on the membrane surface, features a new location for the H1/2 hairpin that satisfies a large set of FRET measurements. The model is compatible with biochemical data and coherent with its function in constituting a TM element in the open-state channel. In addition, from a comprehensive kinetic analysis, we reconciled a series of transient signals reported in the literature with a 7-state model previously reported [26,34] and we revised the elemental steps or processes according to the nature and properties of the intermediates, by

absorption

onset integration

integration

elongation

adjustment

From these analyses, we have identified that the first transition (E1 / E2) as being able to elicit structural changes, while the socalled insertion-step (E5 / E6) [26,34,45], does not evoke major conformational changes. Moreover, the kinetic analysis revealed a simultaneous process involving the unfolding and insertion processes, named herein integration steps, which could be separated into two discrete intermediates, E3 and E4. These are envisioned as the opening of an umbrella-like structureethe anchoring of a vertical central element surrounded by surface-bound peripheral elements, rather than a sequential unfolding into an extended array of a-helices on the membrane surface before the insertion of the hydrophobic elements. In summary, the “umbrella” model for the two-dimensional arrangement of ColE1c on the membrane surface shows a suitable location for the H1/2 hairpin according to FRET measurements and is compatible with its proposed role as a functional entity that constitutes a TM element in the open-state channel. In addition, the 3D-structure of this model harmonizes with an insightful integration mechanism derived from transient kinetics. We cannot say, though, that we have mapped unequivocally the exact location of the secondary elements and configuration of the side-chains. Rather, our 3D-structure must be considered as possessing an overall disposition of the H1eH7 segment on the membrane, in relation to the embedded central H8/9 helical hairpin. Acknowledgements Research reported in this work was supported by the Natural Sciences and Engineering Research Council of Canada (Grant number 105440-2013) to ARM. We thank Tom Keeling for technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.abb.2016.08.007. References [1] A.P. Pugsley, Microbiol. Sci. 1 (1984) 203e205. [2] E. Cascales, S.K. Buchanan, D. Duche, C. Kleanthous, R. Lloubes, K. Postle, M. Riley, S. Slatin, D. Cavard, Microbiol. Mol. Biol. Rev. 71 (2007) 158e229. [3] A. Filloux, R. Voulhoux, B. Ize, F. Gerard, G. Ball, L.F. Wu, Biochimie 84 (2002) 489e497. [4] S.D. Zakharov, W.A. Cramer, Front. Biosci. 9 (2004) 1311e1317. [5] W.A. Cramer, J.B. Heymann, S.L. Schendel, B.N. Deriy, F.S. Cohen, P.A. Elkins, C.V. Stauffacher, Annu. Rev. Biophys. Biomol. Struct. 24 (1995) 611e641. [6] S.D. Zakharov, M. Lindeberg, Y. Griko, Z. Salamon, G. Tollin, F.G. Prendergast, W.A. Cramer, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 4282e4287. [7] Y.K. Shin, C. Levinthal, F. Levinthal, W.L. Hubbell, Science 259 (1993) 960e963. [8] P. Elkins, A. Bunker, W.A. Cramer, C.V. Stauffacher, Structure 5 (1997) 443e458. [9] P.A. Elkins, H.Y. Song, W.A. Cramer, C.V. Stauffacher, Proteins 19 (1994) 150e157. [10] M.W. Parker, J.P. Postma, F. Pattus, A.D. Tucker, D. Tsernoglou, J. Mol. Biol. 224 (1992) 639e657. [11] S.L. Schendel, W.A. Cramer, Protein Sci. 3 (1994) 2272e2279. [12] Y.V. Griko, S.D. Zakharov, W.A. Cramer, J. Mol. Biol. 302 (2000) 941e953. [13] Y. Kim, K. Valentine, S.J. Opella, S.L. Schendel, W.A. Cramer, Protein Sci. 7 (1998) 342e348. [14] S.D. Zakharov, W.A. Cramer, Biochim. Biophys. Acta 1565 (2002) 333e346. [15] C. Lesieur, B. Vecsey-Semjen, L. Abrami, M. Fivaz, G.F. Gisou van der, Mol. Membr. Biol. 14 (1997) 45e64.

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E1 ! E2 ! E3 !E4 ! E5 ! E6 ! E7

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