Hydrodynamic Characterization of the DEAD-box RNA Helicase DbpA

doi:10.1016/j.jmb.2005.10.058

J. Mol. Biol. (2006) 355, 697–707

Hydrodynamic Characterization of the DEAD-box RNA Helicase DbpA Miguel A. Talavera1†, Erin E. Matthews2†, William K. Eliason1, Irit Sagi3 Jimin Wang1, Arnon Henn1 and Enrique M. De La Cruz1* 1

Department of Molecular Biophysics & Biochemistry Yale University, New Haven CT 06520, USA 2 Department of Chemistry Yale University, New Haven CT 06520, USA 3

Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel

The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, sizeexclusion chromatography, analytical ultracentrifugation, chemical crosslinking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 mM and over the temperature range of 4 8C to 22 8C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23 S rRNA and interacts specifically with hairpin 92 of the PTC. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: DbpA; helicase; RNA; analytical ultracontrifugation; bead modeling

Introduction DExD/H-box proteins are ubiquitous enzymes that facilitate RNA structural rearrangements through the disruption of RNA–RNA or RNA–protein contacts in reactions that are coupled to the binding and hydrolysis of ATP.1,2 DEAD-box proteins are essential in all aspects of RNA metabolism including splicing, ribosome biogenesis, RNA degradation, and translation. A robust RNA-activated ATPase activity, high sequence similarities with DNA helicases belonging to both the helicase superfamily-I (SF-I) and the helicase superfamily-II (SF-II), and involvement in cellular processes that require rearrangement of RNA † M.A.T. & E.E.M. contributed equally to this work. Present address: M. A. Talavera, United States Patent and Trademark Office, Remsen Building- 4B71, 400 Dulany Street, Alexandria, VA 22314, USA. Abbreviations used: DbpA, DEAD-box protein A; PTC, peptidyl transferase center. E-mail address of the corresponding author: [email protected]

structures, favor models in which DEAD-box proteins function as ATP-dependent RNA helicases.3–5 DbpA is a w49 kDa DEAD-box RNA protein from Escherichia coli implicated in ribosome biogenesis because it possesses an RNA-dependent ATPase activity that is specifically activated by the peptidyl-transferase center (PTC) of 23 S ribosomal RNA.6 DbpA is part of the SF-II of nucleic acid helicases, and is characterized by a central “helicase core” domain that contains at least seven canonical helicase motifs, including an ATP binding pocket consisting essentially of motif I and the signature DEAD-box sequence in motif II.7 DbpA also possesses a basic carboxyl-terminal domain thought to determine the specificity for ribosomal RNA.8,9 The ability of DbpA to unwind duplex RNA, a process requiring the binding and hydrolysis of ATP, has been shown using bulk biochemical and single-molecule imaging assays.10,11 DbpA requires hairpin 92 of the PTC for unwinding activity of short 10-mer RNA oligonucleotides positioned either up-stream or down-stream of hairpin 92 in

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

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Hydrodynamic Characterization of DbpA

the PTC.11 It was proposed that DbpA performs its “specific” unwinding activity by anchoring its C-terminal extension on hairpin 92, while its core region unwinds a proximate duplex RNA region.12 However, DbpA can also unwind non-specific sequences of long duplex RNA molecules,10 indicating that DbpA is a unique helicase with a core capable of unwinding substrates independent of their sequence and a C-terminal domain capable of imparting specificity. Knowledge of the oligomeric structure of helicases is fundamental for understanding the molecular mechanism of active unwinding. 13–14 Proposed mechanisms for nucleic acid duplex unwinding by canonical DNA helicases implicate nucleotide-dependent single and double-stranded DNA binding affinities13–14 and a minimum of two DNA binding sites. The requirement for multiple binding sites can be fulfilled if an individual monomeric helicase molecule possesses multiple nucleic acid binding sites, and/or if the helicase can self-associate into higher order oligomers, with each subunit possessing a nucleic acid binding site.13–14 To understand how nucleotide-dependent conformational changes within DbpA are coupled to RNA unwinding, an accurate description of the active DbpA structure is essential. We have used analytical ultracentrifugation, size-exclusion chromatography, multi-angle light-scattering and chemical cross-linking to evaluate the state of DbpA, and determine if nucleotide and RNA binding modulate it. Our results favor a mechanism where DbpA functions as a monomeric helicase with two distinct RNA binding sites.

Results Size-exclusion chromatography (SEC) and multi-angle laser light-scattering (MALLS) analysis of DbpA

protein) sphere with a molecular mass of 49,188 Da and a partial specific volume ðv2 Þ of 0.743 cm3 gK1 calculated using:
1=3 3M2 ðv2 C dv 01 Þ (1) RS Z 4pNA where v 1 , the partial specific volume of water, is ˚ (Table 1), smaller than the experimentally 27.9 A determined value. The ratio RS (experimental)/RS (theoretical) is w1.13–1.17, suggesting that DbpA is a non-spherical, asymmetric monomer in solution. The light-scattering profile of DbpA displays a weak angular-dependence (Figure 1(b)). The weight-average molecular mass of DbpA in solution calculated from extrapolation to infinitely small angle (intercept of Figure 1(c)) is 55.1 kDa (Table 1). DpbA with bound MgADP elutes as a single peak with a relative molecular mass of 56.4 kDa (Table 1). Similarly, a DbpA point mutant, where the putative catalytic residue glutamate15 was mutated to glutamine to prevent ATP hydrolysis, behaves as a w53 kDa monomer in the presence of 2 mM MgATP or MgADP (Table 1). Sedimentation equilibrium analysis Sedimentation equilibrium experiments were performed using three DbpA concentrations, three rotor speeds and two temperatures (Figure 2). Global analysis of the data indicates that DbpA in solution behaves as a 49 kDa non-interacting monomer at 4 8C and at 22 8C. No self-association was observed at protein concentrations up to 25 mM in buffer A (see Materials and Methods for buffer composition; Figure 2). Fitting the data sets to a reversible monomer–dimer equilibrium model (equations (13) and(14)) does not significantly improve the fit as indicated by the residuals of the best fits (not shown). Sedimentation velocity analysis

DbpA elutes from a Superdex 75 size-exclusion column as a single peak with a Stokes radius of ˚ (Table 1, Figure 1). The predicted radius of a 32.7 A hydrated (assuming d is 0.3773 g water per 1 g of

The presence of a single sharp boundary (Figure 3(a)) indicates that DbpA sediments as a single, monodisperse species. The continuous sedimentation distribution (c(s) versus s,

Table 1. Summary of DbpA hydrodynamic parameters Species

Method a

DbpA DbpA DbpA

Sphere Gel filtration Sed. velocity

DbpA DbpA DbpA-ADP E154Qd-ATP E154Qd-ADP

Sed. equilibrium MALLS MALLS MALLS MALLS

a b c d

˚) Rs (A

f (10K8 g sK1)

s20,w (10K13 s)

D20,w (10K7 m2 sK1)

f/fo

MW (kDa)

27.9 32.7 31.7

4.60 6.18 5.99

4.6 3.87 3.5

8.85 6.55 6.75

1.0 1.2 1.3 1.4b

– 56.0 49.2c

Calculations for a spherical hydrated protein particle of 49,188 Da. Determined from simulated c(s) distributions.  Calculated using MWZ s020;w NA f =ð1KvrÞ. Single point mutation (E154Q) replacing putative residue involved in hydrolyzing g phosphate group of ATP.15

49.0 55.1 56.4 53.0 53.2

699

Hydrodynamic Characterization of DbpA

Figure 2. Sedimentation equilibrium analysis of DbpA. The lower panels show the equilibrium distribution of DbpA at a loading concentration of (a) 23.8 mM, (b) 15.9 mM, and (c) 8 mM acquired at rotor speeds of 10,000 (green), 18,000 (blue), and 22,000 (red) rpm, 22 8C. The continuous lines represent the best fits of the data to equation (11). The upper panel shows the residuals of the best fits. Note the different ordinate scale of the residuals plot.

dilution. Consequently, the diffusion coefficients are lower at high protein concentrations. The limiting diffusion coefficient ðD020;w Þ is 6.752!10K7 cm2 sK1. There is a strong concentration-dependence of the observed sedimentation coefficient. The high charge density of the DbpA C terminus and asymmetric shape of DbpA may account for this behavior.

Figure 1. SEC/MALLS analysis of DbpA. (a) Stokes radius of DbpA determined by gel-filtration chromatography. (b) Elution profile of DbpA acquired by simultaneous detection of absorbance at 280 nm (- - -), solution refractive index (/), and 908 scattering of 690 nm light (continuous). The MW distribution is calculated for the area between the vertical lines. (c) Angular-dependence of DbpA light-scattering intensity (Zimm plot). The intercept yields the scattering intensity at infinitely small angle and is used to calculate the MW distribution of DbpA.

Figure 3(b)) calculated using numerical solutions of the Lamm equation16 (equation (15)) yields a single symmetrical peak, confirming the presence of a monodisperse species in solution. The best fit of the data gave an s value of 3.37 S for 25 mM DbpA at 22 8C (Figure 3(b)). Small amounts (%1% of total sample) of a rapidly sedimenting species of 6–10 S were observed in samples of the highest concentrations. The sedimentation coefficient of DbpA decreases with concentration (Figure 4), as expected for a nonassociating single particle.17 A limiting sedimentation coefficient ðs020;w Þ of 3.5 S is obtained at infinite

Computational and hydrodynamic modeling of DbpA The Robetta server† constructed ten DbpA models using both comparative modeling and de novo structure prediction methods.18 All generated models consist of two distinct domains: the N-terminal domain, consisting of the helicase core, and the C-terminal domain. By homology modeling, the DbpA helicase core domain (amino acid residues 1–360) is predicted to adopt a structure similar to the helicase core of other DEAD-box proteins including eIF4A,19 mjDEAD, BstDEAD and UAP56, consistent with previous structural homology models.20 The C-terminal region of DbpA (371–457) did not give sequence matches to proteins of known structure. Therefore, a structure prediction was made using the de novo Rosetta fragment insertion methods.21 The predicted C-terminal fold consisted of a b-hairpin and an a-helix. The surface of the predicted C terminus has a positive electrostatic potential due to a cluster of basic amino acid † http://robetta.bakerlab.org/

700

Hydrodynamic Characterization of DbpA

residues, consistent with this domain playing a role in RNA binding.12 Although the predicted structure of the C-terminal domain is a computational model, and should be viewed as such, the compact nature of the predicted fold is consistent with studies showing the C-terminal domain is not susceptible to protease cleavage,20 and allows us to model conformations of DbpA consistent with the hydrodynamic parameters determined experimentally. The major difference amongst the

Figure 3. Sedimentation velocity analysis of DbpA. (a) The absorbance profiles were acquired at 280 nm, a protein concentration of 25 mM, a rotor temperature of 22 8C and a rotor speed 35,000 rpm. For clarity of presentation only every 15th dataset is shown. Measured absorbance distributions are shown as colored lines. Bestfit (red lines) forms of the Lamm equation were obtained using the SEDFIT program. Lower panel shows the residuals of the fits. (b) Calculated distribution c(s) versus s of the DbpA protein, using the sedimentation profiles shown above.

Figure 4. Concentration dependence of the observed sedimentation and diffusion coefficients of DbpA. The s020;w was determined by extrapolation to zero protein concentration, yielding the value 3.5 S.

Figure 5. Structural homology modeling of full-length DbpA illustrating the possible orientations of the de novogenerated C-terminal domain. (a) Structural homology model of DbpA illustrating the possible orientations of the C-terminal domain. The ribbons diagrams46 are colored at the helicase core (residues 1–371 are silver; P-loop motif, residues 47–54 are magenta; DEAD box, residues 151–156 are cyan; SAT motif, residues 184–186 are yellow; motif VI, residues 325–335 are gold) and at the C-terminal domain. Residues 372–456 are colored red (model 1), magenta (model 2), yellow-green (model 3), cyan (model 5), green (model 7), and blue (model 9). (b) Derived models consistent with hydrodynamic measurements (sedimentation coefficient and diffusion coefficients).

701

Hydrodynamic Characterization of DbpA

Figure 6. Hydrodynamic parameters of structural models. Relationship between the s020;w and D020;w of the predicted structural models (C) and the experimentally extrapolated value (:). Values are summarized in Table 2.

models is a variation in the relative orientations of the N and C-terminal domains (Figure 5). The models with calculated hydrodynamic coefficient values most similar to the experimental sedimentation and diffusion coefficients (models 1, 7 and 9) predict the C-terminal domain and helicase core are brought into close proximity by potential hydrophobic contacts at the core domain surface. Calculations using the remaining models predict hydrodynamic coefficients that vary from those measured experimentally (Figure 6, Table 2). However, all models may account for the experimental data if conformational dynamics involving changes in the relative orientations of the helicase core and C-terminal domains are taken into account. Such conformational fluctuations seem likely given the presumed flexible element linking the two domains. Determination of DbpA–RNA stoichiometry by electromobility shift assay (EMSA) and ATPase activity The stoichiometry of the DbpA–RNA complex was determined using an EMSA by titrating excess PTC-RNA (1.1 mM) with DbpA (Figure 7). The PTCRNA concentration was w1000-fold above the Table 2. Comparison of experimental hydrodynamic coefficients with theoretical values derived from hydrodynamic modeling Species DbpA Model 1 Model 2 Model 3 Model 4 Model 5 Model 6 Model 7 Model 8 Model 9 Model 10

s20,w (S)

D20,w (10K7 cm2 sK1)

3.497G0.079 3.517 3.551 3.391 3.478 3.452 3.554 3.527 3.547 3.506 3.541

6.752G0.135 6.742 6.808 6.500 6.667 6.616 6.813 6.760 6.799 6.721 6.788

Figure 7. Gel electrophoresis mobility-shift assay of DbpA binding to 153-mer PTC RNA. DbpA was incubated with 1.1 mM RNA and resolved by electrophoresis in a native 8% polyacrylamide gel. The gel was stained in an ethidium bromide solution. The protein to RNA ratios are (left to right): 0 (i.e. RNA alone), 0.25, 0.49, 0.74, 0.98, 1.27, 1.47, 1.71, 1.96 and 2.24. Lower panel shows fraction of bound (:) and free (C) RNA as a function of the protein/ RNA molar ratio.

binding affinity (Kd w0.5 nM22), ensuring that all of the added protein binds. A slower migrating species, attributed to the DbpA–RNA complex, formed in a [DbpA]-dependent manner (Figure 7(a)). The fraction of bound RNA increased in a linear fashion until reaching the “titration breakpoint” (Figure 7(b)). The best fit of the data yields a stoichiometry of 1.17 DbpA/PTC-RNA, consistent with reported stoichiometric measurements of the complex.22 The PTC-RNA-activated ATPase activity of DbpA is linearly dependent on protein concentration up to concentrations equivalent to the total RNA present (data not shown), consistent with a 1:1 stoichiometry during steady-state cycling.3 Chemical crosslinking EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and DMS (dimethyl suberimidate$2 HCl) were assayed for their ability to chemically crosslink DbpA. Positive controls demonstrate that the crosslinking efficiency is high for BSA23,24 (data not shown) and myosin V (data not shown), a protein known to form a dimer.25,26 No crosslinked DbpA oligomers were detected using the

702

Figure 8. EDC crosslinking of DbpA in the presence and absence of 153mer PTC RNA. DbpA (5 mM) was incubated without (top) or with 10 mM RNA (bottom) for 1 h at room temperature in buffer A. Crosslinking was initiated by the addition of the indicated EDC concentrations and allowed to proceed for 1 h before quenching with excess Tris. The products were then analyzed by SDS-PAGE.

same crosslinking protocol (Figure 8). Inclusion of excess MgADP or the ATP transition-state analogues BeFx:ADP and vanadium:ADP (a fraction bound of w0.8 would be estimated if we assume these analogs bind with affinities comparable to MgATP27) had no effect (data not shown), consistent with DbpA existing as a non-interacting monomer in solution independent of the bound nucleotide and PTC-RNA. ATP-induced dissociation of actomyosin (muscle subfragment 1) complex as assayed by light-scattering28 confirmed that the nucleotide analogues were active (data not shown). Similarly, excess PTC-RNA in the presence and absence of nucleotides had no effect (Figure 8).

Discussion Conformation of DbpA in solution DbpA in solution behaves as a non-associating monomer at concentrations %25 mM. Size-exclusion chromatography, sedimentation equilibrium, and sedimentation velocity show that no significant DbpA–DbpA interactions occur, since the RS and molecular mass values are consistent with a w49 kDa monomeric protein. In addition, chemicalcrosslinking analysis with reagents of different lengths (e.g. EDC is a zero length crosslinker; DMS

Hydrodynamic Characterization of DbpA

˚ ) could not detect DbpA has a length of 10 A oligomerization. Therefore, these complementary methods established that DbpA exists as a stable monomer up to a concentration of at least 25 mM over the temperature range of 4 8C to 22 8C, suggesting a predominantly monomeric structure of DbpA at assay and physiological concentrations.6,27 The three-dimensional structure of DbpA is not yet available. However, our comparative modeling predicts that DbpA adopts a structure similar to the DEAD-box protein eIF4A, whose architecture is similar to the other well-characterized DNA and RNA helicases PcrA, Rep and HCV NS3.4,7 These helicases fold into a globular core structure consisting of two large domains separated by a cleft where the nucleotide and nucleic acid bind. The predicted structure of the DbpA helicase core is globular, similar to these other helicases, suggesting that the non-spherical shape of DbpA arises from the additional mass and volume of the C-terminal domain as predicted from the plausible structural models (Figure 5). The structural models with hydrodynamic coefficients consistent with the experimentally determined values predict that the C-terminal domain and helicase core interact through hydrophobic contacts (Figure 5). This interaction may influence the dynamics of the core domain and account for the weak ATP binding affinity27 and low intrinsic ATPase activity of DbpA in the absence of RNA,27 and for the large and specific activation of the ATPase activity by PTC-RNA.3 It is likely that PTC-RNA binding to the C-terminal domain changes the relative core and C terminus orientations and releases the C-terminal domain from the core surface (Figure 5), which may allow the helicase core to bind and hydrolyze ATP. Monomer is the functional form of DbpA The hydrodynamic characterization of DbpA in the presence and absence of ADP or ATP analogues demonstrates that DbpA is a monomer in solution that does not undergo nucleotide-induced oligomerization. In addition, the stoichiometric steadystate ATPase activation and lack of chemical crosslinking in the presence of PTC-RNA and ATP analogues suggests that DbpA is an active monomer during PTC-RNA-activated steady-state ATPase cycling. DbpA is therefore likely to possess two distinct RNA binding sites. One site presumably lies in the helicase core domain. The highly basic nature of the C terminus suggests that this domain also binds RNA as suggested.12,29 Although there is no direct evidence for PTCRNA-induced oligomerization from our experiments, large double-stranded RNA substrates appear to promote such activity.10 Our binding and crosslinking determinations did not reveal any occurrence of oligomerization, thus the appearance of oligomers with larger double-stranded RNA substrates may be conditional with the use of such substrates and it is therefore possible that DbpA can unwind as a monomer or oligomer depending on

703

Hydrodynamic Characterization of DbpA

the secondary and/or tertiary structural complexity of the RNA substrate.

during in vivo catalysis would prevent rapid reannealing of the RNA duplex and recycle DbpA.

Implications for PTC-RNA unwinding activity Helicase-catalyzed RNA unwinding is a multistep process that may involve nucleotidedependent single-stranded and double-stranded RNA binding affinities.30 Although the affinities of the DbpA core for single and double-stranded RNA have not been determined, quantitative studies have indicated that the relative affinities of DNA helicases for single and double-stranded DNA are not DNA sequence-dependent but are modulated by the nucleotide bound to the helicase core.31,32 The ADP bound state of DNA helicases binds more tightly to double-stranded than single-stranded DNA, and with bound ATP and ATP analogues, single-stranded DNA binding is preferred.14 Given the high sequence homology and predicted fold of the core domain, we hypothesize that the core binds single and double-stranded RNA segments of PTC-RNA with different nucleotidedependent affinities, similar to characterized DNA helicases. It is likely that the C-terminal domain binds specifically to hairpin 92 of PTC-RNA in a nucleotide-independent manner, as it lacks any nucleotide binding motifs. We favor the following simplified model for nucleotide-dependent DbpA-catalyzed unwinding of PTC-RNA: ATP binding favors binding of a double-stranded RNA segment (presumably H89 and/or H9122) to the helicase core. Hydrolysis of bound ATP and release of Pi modulates the RNA affinity of the core, promoting single-stranded RNA binding, and therefore facilitating duplex RNA unwinding. We implicate a single ATP turnover per unwinding event but multiple ATP molecules may be required. Although DbpA is capable of unwinding long RNA duplexes and such activity may require the action of DbpA oligomers, most naturally occurring RNA molecules, including 23 S rRNA, have alternations of helical and non-helical conformational angles, rendering a more complex substrate for unwinding than one consisting only of a classic A-form RNA duplex. Such a complex substrate may not allow the protein to progress along the RNA efficiently, and DbpA may act as a rather poor helicase on the PTC-RNA. Therefore, as suggested for most DExD\H-box proteins, DbpA may function as an unwindase or RNA chaperone-like ATPase33,34 associated with the assembly of the 23 S rRNA, where the RNA specificity is dictated by the C-terminal domain of the protein. Exactly how DbpA releases the unwound substrate and dissociates from the 23 S ribosomal RNA is not clear. If DbpA indeed participates in the biogenesis of bacterial ribosomes, then release of the unwound PTC-RNA substrate may involve a “hand-off” mechanism to ribosomal proteins or regions of the 23 S rRNA. A hand-off mechanism

Materials and Methods Reagents All chemicals were the highest purity commercially available. ATP (99C% purity as assayed by HPLC, data not shown) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). ADP (99C% purity as assayed by HPLC, data not shown), RNase A, myoglobin, ovalbumin, albumin, and alcohol dehydrogenase were from Sigma (St. Louis, MO). ATP and ADP concentrations were determined by absorbance at 259 nm using an 3259 of 15,400 MK1 cmK1. A molar equivalent of MgCl2 was added to ATP and ADP immediately before use. Unless stated otherwise, all experimental measurements were made in buffer A (100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 20 mM Hepes, pH adjusted to 7.5 at the experimental temperatures). DbpA has an overall small charge of 0.75 at this pH (calculated with SEDNTERP). Protein purification DbpA was over-expressed in E. coli strain BL21(DE3) pLys-S and purified by ion-exchange (DEAE-Sepharose and SP-Sepharose, 10 cm!2.5 cm, Pharmacia) and sizeexclusion (Superdex 75-HR, 1.6 cm!60 cm, Pharmacia) chromatography.27 The DbpA concentration was determined by absorption at 280 nm in buffer A using an 3280 of 25,200 MK1 cmK1. Experiments were performed within five days of purification. Preparation of the PTC ribosomal RNA The DNA encoding the PTC rRNA was constructed using recursive PCR35 with oligonucleotides spanning nucleotides 2454–2606 of the E. coli 23 S rRNA. To facilitate cloning and transcription, the coding region was preceded by an EcoRI site and the phage T7 RNA polymerase promoter, and followed by a BamHI site. Using the EcoRI and BamHI sites, the coding sequence was inserted into a pUC19 vector. Plasmid was amplified in E. coli (DH5a), purified with Nucleobond-AX (BD Biosciences Clontech), digested with BsaI, extracted with one volume of Tris-buffered (pH 7.9) saturated phenol, then one volume of a 1:1 mixture of Tris buffered (pH 7.9) saturated phenol/chloroform, and finally precipitated with absolute ethanol. RNA was made by in vitro transcription using the T7 promoter built into the plasmid template.36 Transcripts were purified on 8 M urea/PAGE, passively eluted into RNase-free water, and desalted by successive concentrations and dilutions with RNase-free water on an Amicon Ultrafiltration (Millipore) cell equipped with 10,000 Da molecular mass cutoff membranes. Purified RNA was quantified by absorbance at 260 nm and analyzed by 8 M urea/PAGE to assess quantity and integrity of the transcript. RNA samples were annealed by heating to 75–80 8C for 5 min and cooled to room temperature in an aluminum block before performing experiments.

704

Hydrodynamic Characterization of DbpA

the weight-average molecular mass (MW) of the solute particle according to:40

Steady-state ATPase activity The steady-state ATPase activity of DbpA was measured at 37 8C in buffer A supplemented with 2 mM MgATP using the malachite green Pi detection assay.37 The RNA-activated ATPase activity was measured using the 153-PTC RNA fragment. Size-exclusion chromatography DbpA was filtered over Superdex-75 (Hiload 16/60, 1.6 cm!60 cm) in buffer A at a flow rate of 1.5 ml minK1. Elution was monitored by absorbance at 280 nm. The column was calibrated using proteins of known Stokes ˚, radii (RS) and molecular masses (M): RNase A (RSZ16.4 A ˚ , 16.9 kDa), ovalbumin 13.7 kDa), myoglobin (RSZ20.2 A ˚ , 43.5 kDa), albumin (RSZ35.5 A ˚ , 67 kDa), and (RSZ30.5 A ˚ , 150 kDa). The partition alcohol dehydrogenase (RSZ45 A coefficient (Kav), was calculated using:
 Ve KVo Kav Z (2) Vt KVo where Ve is the elution volume of the sample, Vo is the excluded, or void, volume of the column and Vt is the total volume of the column. An excluded volume (Vo) of 120 ml and a total volume (Vt) of 340 ml were measured with blue dextran and thymidine, respectively. The Stokes radius pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi of DbpA was determined from the plot of Klog Kav versus RS of the calibration standards.38,39 The frictional (f) and diffusion (D) coefficients were calculated from the Stokes radius (RS) according to Stokes’ law (equation (3)) and the Stokes–Einstein relation (equation (4)), respectively: f Z 6phRS DZ

RT NA f

(3) (4)

where h is the solvent viscosity, R is the universal gas constant (8.314!107 erg/mol K), and NA is Avogadro’s number. Multi-angle laser light-scattering Multi-angle laser light-scattering experiments were performed in buffer A at 22 8C. Light-scattering data were collected on a DAWN EOS (Wyatt Technology) coupled to an OPTILAB DSP (Wyatt Technology) interferometric refractometer. Samples (150 ml of 60 mM DbpA) were gel-filtered over Superdex-200 (HR-10/30) at a flow rate of 0.5 ml minK1. Multi-angle laser lightscattering (690 nm), absorbance (280 nm) and the refractive index were monitored after elution. Monomeric bovine serum albumin (Sigma, A1900) was dissolved in buffer A at 5–6 mg mlK1 and gel-filtered prior to DbpA sample analysis. Data collected from the peak corresponding to monomeric bovine serum albumin were used to normalize the light-scattering intensities for all detector angles and to estimate the protein concentrationdependent change in solution refractive index value (dn/dc) for determination of the DbpA concentration. Light-scattering intensities were detected at multiple angles (qZ42.8, 51.5, 60, 69.3, 79.7, 90, 100.3, 110.7, 121.2, 132.2 and 142.58) relative to the incident beam. Analysis of light-scattering data (ASTRA for Windows version 4.73, Wyatt Technology) was based on the Zimm formalism of the Rayleigh–Debye–Gans model,40 which relates the light-scattering intensity at angle q (Rq ) to

K c 1 C 2A2 c Z Rq MWPðqÞ

(5)

where c is the solute concentration (in g/ml) as measured by the on-line UV and refractive index detectors, A2 is the second virial coefficient (ml mol gK2) that accounts for solvent–solute interactions (which are small and can be ignored at the protein concentrations encountered in chromatographic analysis), and K* is a physical constant defined as: K Z

4p2 n2o ðdn=dcÞ2 NA l4o

(6)

where no is the refractive index of the solvent (1.33 for aqueous), and lo is the wavelength of the incident light employed in the experiment (690 nm). The function P(q) describes the angular dependence of scattered light.40 Particles smaller than the incident light wavelengths (lo) do not show an angular-dependence and P(q) equals 1. For larger scattering particles, with dimensions comparable to the employed wavelength, the interference effects produced by the scattered radiation from different points on the same particle show an angular-dependence of the scattered light intensity (Rq). The reciprocal P(q)K1 can be expressed as a power series:

 1 16p2 2 2 q Z1C C/ (7) hrg isin PðqÞ 2 3l2 illustrating that P(q) is independent of the shape of the scattering particles and depends only on the particle rootmean-square radius(hrg2i, radius of gyration). As sin2(q/2) approaches zero, P(q)K1 approaches 1 and equation (5) simplifies to: K c 1 Z q/0 RðqÞ MW

lim

(8)

Consequently, plotting the quotient K*c/Rq versus sin2(q/2) (Zimm plot, Figure 1(c)) and extrapolating to the ordinate intercept (i.e. sin2(q/2)Z0) yields the shapeindependent MW. Sedimentation equilibrium Sedimentation equilibrium experiments were performed at 4 8C and 22 8C using a Beckman Optima XL-I analytical ultracentrifuge. DbpA in buffer A 0 (100 mM NaCl, 1 mM DTT, 20 mM Hepes, pH 7.5) at three different concentrations (13, 18, and 25 mM) were centrifuged at three different velocities (9000 rpm, 12,000 rpm, and 16,000 rpm at 4 8C, and 10,000 rpm, 18,000 rpm, and 22,000 rpm at 22 8C) in an AN 60-Ti 4-hole rotor equipped with a six channel Epon-charcoal filled centerpiece. Absorbance at 280 nm was used to monitor the radial migration of DbpA. Attainment of equilibrium at each speed was confirmed by comparing three successive scans at 1 h intervals with the program WinMatch (available from the National Analytical Ultracentrifugation Facility website†).  of DbpA was calculated The partial specific volume ðvÞ from the amino acid composition and corrected for temperature using SEDNTERP (available from the † http://vm.uconn.edu/~wwwbiotc/uaf.html

705

Hydrodynamic Characterization of DbpA

RASMB web site†). Correction of v to the experimental temperature ðvT Þ was based on: vT Z v25 C 4:25 !10K4 ðTK25Þ

(9)

where v25 is the predicted value at 25 8C. Values of 0.735 and 0.743 cm3/g were obtained for DbpA at 4 8C and 22 8C, respectively. Solvent density (r) was approximated as 1 g/ml. At equilibrium, sedimentation and diffusion forces are balanced, and the total concentration of the macromolecule in the rotor cell (ctotal) can be expressed as:41 n n X X ctotal Z ci ðrÞ Z K1;i c1 ðrÞqðiÞ (10) iZ1

iZ1

where q(i) is the state of the ith species, K1,i, the equilibrium constant for the association of monomer to the q(i)-mer, and n is the number of species present. For a single homogenous species the concentration at radius r (c(r)) can be expressed as: " # n X s 2 2 cðrÞ Z cðr0 Þ exp ðr Kr0 ÞK2B ci ðrÞ (11) 2 iZ1 where c0 is the macromolecule concentration at reference radius r0, B is the colligative second virial coefficient, and s is the reduced molecular mass defined as: 2  MWð1KvrÞu (12) RT where MW is the amino acid sequence molecular mass, v is the partial specific volume of the solute particle in cm3/g, r is the solvent density in g/cm3, u is the angular velocity (2p rpm/60) in rad/s, and T is the absolute temperature in Kelvin. For a reversible monomer (c1):dimer (c2) equilibrium, the association equilibrium constant for dimerization (Ka) is: c Ka Z 2 2 (13) ðc1 Þ

sZ

where c2 is the dimer concentration, and equation (11) can be expressed, assuming ideality, as: hs i hs i ctotal ðrÞ Z c1;r0 exp ðr2 Kr20 Þ C c2;r0 exp ðr2 Kr20 Þ (14) 2 2 In data analysis with WINNONLIN (available from the RASMB website†), non-linear least-squares regression was used to optimize parameters in a minimum of nine data sets (three concentrations, three speeds). The value of s1 was fixed to correspond to the calculated reduced molecular mass of the monomer determined from sequence analysis and MALDI mass spectrometry. Sedimentation velocity Sedimentation velocity experiments were performed at 4 8C and 22 8C using Epon charcoal-filled 12 mm double-sector centerpieces. DbpA in buffer A 0 (440 ml at 5, 15, 20, and 25 mM) was centrifuged at 35,000 rpm. Radial absorption scans (lZ280 nm) were acquired every 5 min until completion of sedimentation. The program SEDFIT16 (available from the RASMB web site†) was used to model the sedimentation profiles using the integrated Lamm equation solutions: ð Aðr; tÞ Z c0 ðsÞcðs; D; r; tÞds C 3 C d (15)

† http://www.bbri.org/rasmb/rasmb.html

where A(r,t) is the absorbance at radius r and time t, s and D are sedimentation and diffusion coefficients, c0 is the loading concentration of the macromolecule, c is the sedimentation profile (i.e. boundary), and 3 and d are signal offsets (the first term accounts for systematic errors such as flaws in optics and time-dependent vibrations and the second term accounts for random errors). Sedimentation coefficient values between 0.5 S and 10 S were resolved to 0.05 S using maximum entropy regularization with a 0.95 confidence level. The diffusion coefficient was calculated using the Svedberg equation:42 DZ

sRT  MWð1KvrÞ

(16)

Intrinsic sedimentation and diffusion coefficients (s020;w and D020;w , corrected to water at 20 8C, were calculated from the experimental s and D values40 according to:

  T;B h 1Kvr (17) s020;w Z sobs 20;w  20;w hT;B 1Kvr D020;w Z Dobs




h20;w hT;B

 (18)

where s020;w and D020;w are the coefficients in the standard solvent of water at 20 8C; sobs and Dobs are the measured coefficients in the experimental solvent at the experimental temperature (T); hT,B and h20,w are the buffer viscosities at the experimental temperature and water at 20 8C, respectively; r20,w is the water density at 20 8C, and rT,B is the buffer density at the experimental temperature. Gel electrophoresis mobility-shift assays DbpA binding to PTC-RNA was assayed by gel electrophoretic mobility-shift assays. Annealed RNA was mixed with a range of DbpA concentrations in buffer A to a final reaction volume of 50 ml. Binding reactions were equilibrated for 1–1.5 h at room temperature and partitioned by electrophoresis through an 8% (w/v) polyacrylamide gel (37.5:1 (w/w) acrylamide:N-N0-methylene-bis-acrylamide ratio in 66 mM Hepes/33 mM Tris– HCl and 2.5 mM MgCl2). After mixing the components for the unpolymerized acrylamide solution, the final pH was 7.5. The gel was run at constant power (6 W) for 90 min at room temperature, followed by staining with 1 mg mlK1 ethidium bromide solution for 30 min. Free and bound RNA was visualized with a UV-transilluminator and quantified using the NIH image software‡. Binding stoichiometries were obtained by fitting the band intensities to equation (19): IðrÞ Z Io C ðIN KIo Þ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 0 1 r 2 Kd Kd r C C n K r C C n K4rn ½DbpAo ½DbpAo B C C !B @ A 2n ð19Þ where I(r) is the band intensity (I) as a function of the [RNA]/[DbpA] ratio (r), Io is the initial intensity in the absence of DbpA, IN is the band intensity at infinitely high r (i.e. saturating [RNA]/[DbpA]), Kd is the apparent dissociation equilibrium constant of the DbpA–PTC ‡ http://rsb.info.nih.gov/nih-image/

706 complex, [DbpA]o is the total DbpA concentration, and n is the stoichiometry of DbpA binding to PTC-RNA. The stoichiometry (n), initial (Io) and final (IN) intensities were unconstrained when fitting. Kd was constrained to the published value of 0.44 nM.22 The titration curves define the stoichiometry better than they define the affinity because [RNA]o[Kd. Chemical cross-linking Chemical cross-linking was performed with EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and DMS (dimethyl suberimidate$2HCl) (Pierce). Varying concentrations of a freshly prepared EDC or DMS stock solutions (250 mM) in buffer A were incubated with DbpA (5 mM) in the presence or absence of 10 mM PTC-RNA for 60 min at room temperature. Samples were quenched by threefold dilution with Trisloading buffer, resolved by SDS-PAGE (8% acrylamide) and stained with Coomassie brilliant blue. Structure prediction, modeling, and structural superposition The Robetta server18 was used to predict the secondary and tertiary structures of DbpA. The server uses a fully automated structure prediction procedure based on comparative modeling to homologous sequences of known structure and de novo structure prediction algorithms to generate structural models. Calculations of electrostatic potential mapped to the accessible surface of DbpA models were performed using the program GRASP.43 The program maps a molecule onto a three-dimensional cubic grid, and calculates the electrostatic potential at each grid point using a finite difference solution to the non-linear Poisson–Boltzmann equation. The accessible surface area of the model was defined as the surface mapped out by the center of a ˚ rolled around the van der Waals probe of radius 1.4 A surface of the protein model. An ionic strength of 130 mM (100 mM NaCl and 5 mM MgCl2) and an ionic radius of ˚ were used. The dielectric constant (relative to a 2.0 A vacuum) was 2 for the protein interior and 80 for the surrounding solvent. Charges were assigned to the ionized groups of Asp and Glu (K1) and Lys and Arg (C1). Histidine and termini residues were neutral. Hydrodynamic bead modeling The program pdb to bead (kindly provided by Dr Walter F. Stafford III, Boston Biomedical Research Institute) was used to transform the atomic coordinates of the generated DbpA models into bead models comprising spheres of a ˚ ). The radius dependent on the resolution chosen (3–3.5 A anhydrous sedimentation and diffusion coefficients for the bead model were calculated using the program HYDRO.44,45 To compare the HYDRO output with the values of the measured coefficients, a correction was made for the hydration of the protein using a value of 0.3773 g water/1 g of protein calculated from SEDNTERP.

Acknowledgements We are grateful to Dr Don Engelman (MB&B, Yale University) for use of the analytical ultracentrifuge

Hydrodynamic Characterization of DbpA

and encouragement, and to Dr Walter F. Stafford III (Boston Biomedical Research Institute) for providing the pdb to bead program. This work was supported by a Hellman Family Fellowship (to E.M.D.L.C.), and by grants from the National Science Foundation (MCB-0216834 to E.M.D.L.C.), the American Heart Association (0235203N to E.M.D.L.C.), and the NIH (GM054160 to Donald M. Engelman). M.A.T. was supported by an NIH NRSA pre-doctoral fellowship (GM6460-02). A.H. was supported by an American Heart Association post-doctoral fellowship.

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Edited by D. E. Draper (Received 16 July 2005; received in revised form 15 October 2005; accepted 19 October 2005) Available online 10 November 2005