Studying assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy

Accepted Manuscript Studying the assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy Cecilia Pinto, Deni Mance, Manon Julien, Mark Daniels, Markus Weingarth, Marc Baldus PII: DOI: Reference:

S1047-8477(17)30211-3 https://doi.org/10.1016/j.jsb.2017.11.015 YJSBI 7132

To appear in:

Journal of Structural Biology

Received Date: Revised Date: Accepted Date:

16 August 2017 24 November 2017 28 November 2017

Please cite this article as: Pinto, C., Mance, D., Julien, M., Daniels, M., Weingarth, M., Baldus, M., Studying the assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy, Journal of Structural Biology (2017), doi: https://doi.org/10.1016/j.jsb.2017.11.015

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Studying the assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy

Cecilia Pinto, Deni Mance, Manon Julien1, Mark Daniels, Markus Weingarth, and Marc Baldus* NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

*corresponding author: [email protected]

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Present address: Laboratory of Structural Biology and Radiobiology, Institute for Integrative Biology of the Cell (CEA, CNRS, University Paris South), University Paris-Saclay, Bât 144, CEA Saclay, F-91191 Gif-sur-Yvette Cedex, France.

Revised for J. Struct. Biol. Special issue: Solid-state NMR of biological assemblies Editors: Tatyana Polenova, Guido Pintacuda, Amir Goldbourt

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Abstract Significant progress has been made in obtaining a structural insight into the assembly of the β-barrel assembly machinery complex (BAM). These crystallography and electron microscopy studies used detergent as a membrane mimetic and revealed structural variations in the central domain, BamA, as well as the lipoprotein BamC. We have used cellular solid-state NMR spectroscopy to examine the entire BamABCDE complex in native outer membranes and obtained data on the BamCDE subcomplex in outer membranes, in addition to synthetic bilayers. To reduce spectral crowding, we utilized proton-detected experiments and employed amino-acid specific isotope-labelling in (13C,

13

C) correlation experiments. Taken together, the

results provide insight into the overall fold and assembly of the BAM complex in native membranes, in particular regarding the structural flexibility of BamC in the absence of the core unit BamA.

Keywords Solid-state NMR, BAM, MAS, E.coli, Membrane protein complex Abbreviations 1 H detection – Proton detection BAM – β-barrel assembly complex DLPC – 1,2-dilauroyl-sn-glycero-3-phosphocholine E. coli – Escherichia coli LPR – Lipid-to-protein ratio OMP – Outer membrane protein OM – Outer membrane POTRA domains – Polypeptide Transport Associated domains ssNMR – solid-state NMR spectroscopy

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Introduction Solid-state NMR (ssNMR) represents one of the few structural methods that can probe membrane protein assemblies at an atomic level (Baker and Baldus, 2014; Herzfeld and Lansing, 2002; Hong et al., 2012; Kaplan et al., 2016b; Marassi and Opella, 1998; Ullrich and Glaubitz, 2013; Wang and Ladizhansky, 2014; Zhou and Cross, 2013). In addition to the well-established use of synthetic bilayer settings, cellular ssNMR concepts have been developed in the recent years that permit extension of such studies to cellular preparations (Baker et al., 2015a; 2015b; Fu et al., 2011; Jacso et al., 2012; Kaplan et al., 2015; 2016a; Renault et al., 2012a; 2012b; Yamamoto et al., 2015). In parallel, great progress has been made to improve ssNMR sensitivity using Dynamic Nuclear Polarization (Kaplan et al., 2016b; Ni et al., 2013) or proton (1H) detection schemes (Andreas et al., 2015; Asami and Reif, 2013; Fricke et al., 2017; Ishii and Tycko, 2000; Weingarth et al., 2014), together with the development of dedicated isotope-labelling schemes that are compatible with the study of large proteins in their natural membrane environment (Baker et al., 2017; 2015a; Mance et al., 2015; Medeiros Silva et al., 2016; Sinnige et al., 2014a). These developments provide novel opportunities for atomic-level studies in a variety of systems, such as β-barrel proteins inserted into the bacterial outer membrane (OMPs) (Renault et al., 2012a), or proteins embedded in the inner membrane of bacteria such as ion and proton channels (Medeiros Silva et al., 2016; Miao et al., 2012; Visscher et al., 2017), retinal proteins (Ward et al., 2015) and electron transport proteins (Yamamoto et al., 2015). More recently, such approaches could also be extended to study, for example, protein complexes that span the bacterial cell envelope, such as the type-4 secretion system (Kaplan et al., 2015), as well as the epidermal growth factor receptor in a mammalian cell setting (Kaplan et al., 2016a). Here we demonstrate the use of such methods to study the assembly of the Escherichia coli (E. coli) β-barrel assembly machinery (BAM) in synthetic lipids, in addition to its natural membrane setting. This molecular machine coordinates the integration of OMPs into the highly asymmetric and complex

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environment of the cellular outer membrane (OM) (Bos et al., 2007; Noinaj et al., 2015). These precursors are recognized and targeted to the BAM complex via dedicated chaperones as they emerge from the Sec translocase. Upon correct folding and insertion these OMPs go on to play roles in a variety of physiological, pathogenic and drug resistance functions (Bos et al., 2007). Structures of the BAM complex in detergent (Bakelar et al., 2016; Gu et al., 2016; L. Han et al., 2016; Iadanza et al., 2016) highlight the complexity of the arrangement of the five proteins within the complex. BamA, itself a β-barrel protein, is at the core of this machinery with five soluble polypeptide transport associated domains (POTRA, in the following referred to as P1 to P5) that serve as a scaffold for the four lipid anchored proteins, BamB, BamC, BamD and BamE (Fig. 1A). The crystal structures however reveal additional intricacies within this basic assembly, namely the existence of two seemingly independent populations, either containing or lacking the BamB protein, which exhibit distinct conformations of the central component, BamA (Fig. 1B and C). Furthermore, BamC (Fig. 1D in red), a protein previously described as a non-canonical lipoprotein that would present its folded domains on the extracellular surface (Webb et al., 2012) was shown to interact, albeit weakly, with the POTRA domains of BamA in this detergent environment (Gu et al., 2016; Noinaj et al., 2015).

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Figure 1. Bam complex and ssNMR samples A. β-barrel assembly complex members: BamB (cyan), BamA (green), BamC (red), BamD (blue) and BamE (orange). Structures of the individual components are taken from the x-ray structure PDB 5D0O (B), except for BamC that is only present in PDB 5D0Q (C). B and C. Detergent solubilized BAM complex structures obtained by x-ray crystallography, PDB 5D0O and 5D0Q respectively, that highlight different conformations of the BamA protein (green) and the absence of the BamC (red) folded domains (B) and the BamB (cyan) protein (C). D. Comparison of the BamCDE subcomplex structure from the x-ray structures depicted in B and C. E, F and G. Schematic representation of the ssNMR samples measured and presented throughout, BamCDE labeled in complex with unlabeled BamAP4P5 in DLPC liposomes (E), BamCDE labeled in E. coli outer membranes (F) and the complete 200 kDa BAM complex in E. coli outer membranes (G).

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Previously, our group has applied both solution-state and ssNMR to study the essential core component of the complex, BamA (Renault et al., 2011; Sinnige et al., 2014a; 2015b; 2014b). These studies have shown how the BamA β-barrel is resilient and can accommodate membrane bilayers of varying hydrophobic thicknesses and that the POTRA domains, in the absence of binding partners, do not display fast global motion in proteoliposomes (Sinnige et al., 2014b). We have also identified within P5 a region of local conformational exchange (Sinnige et al., 2015b) that could be involved in driving protein folding in an energy deficient environment, such as the OM. In the following we present ssNMR results obtained on the entire BamABCDE complex, in the bacterial OM (Fig. 1G), and data obtained on the BamCDE subcomplex in the OM (Fig. 1F) as well as reconstituted into synthetic bilayers (Fig. 1E). In studies of the BamAP4P5-BamCDE complex, we applied a combination of specific amino acid labelling (AVLTMS) and fractional deuteration (Mance et al., 2015; Nand et al., 2012) to reduce spectral crowding and to enhance the spectral dispersion. Taken together these results provide insight into the fold and assembly of the BAM complex in native membranes.

Material and methods Sample preparation Isotope labeled variants of the BamAP4P5-BamCDE co-complex in synthetic liposomes were prepared as follows: Unlabeled BamAP4P5 was prepared as described previously (Sinnige et al., 2015a). For expression of the isotope labeled BamCDE complex, E.coli BL21 star cells, co-transformed with the plasmids for BamCD (pSK46) and BamE (pBamE-His) (Hagan and Kahne, 2011), were grown in minimum M9 medium. For the uniformly

13

C,

15

N-labeled complex,

13

C glucose and

15

N

NH4Cl were added to the medium and protein expression was induced with

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0.5 mM IPTG upon reaching an optical density of ~0.6. For the specifically AVLMTS amino-acid labeled BamCDE subcomplex, cultures were grown in unlabeled medium to an optical density of ~0.6 whereby they were induced with 0.5 mM IPTG for approximately 30 minutes prior to the addition of

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C,

15

N-labeled alanine, valine, leucine, methionine, threonine and serine at a

final concentration of 200 mg/L. In both instances the cultures grew for an additional 4 hours at 37°C after which they were harvested and stored as a pellet. E. coli cells containing over-expressed BamCDE subcomplex were thawed on ice with 10 mM Tris-HCl pH 8, lysozyme and 1 µL DNase I/L of culture. Cell lysis was achieved by using a French press. Unbroken or large debris were removed by a 20 min centrifugation at 4 000xg, 4°C. Subsequently, cellular membranes were harvested by ultracentrifugation at 60 000xg for 1h at 4°C (Beckman Coulter Optima L-90K ultracentrifuge with a SW32 Ti rotor) before solubilization with 50 mM Tris-HCl pH 8, 150 mM NaCl, 1% n-Dodecyl β-D-maltoside (DDM), 10 mM imidazole and protease inhibitors. After solubilization (2h, 4°C) the sample was ultracentrifuged for 1h at 60 000xg and 4°C. The resulting supernatant was applied to Ni-NTA agarose beads (Qiagen) pre-equilibrated with 50 mM Tris-HCl pH 8, 150 mM NaCl, 0.03% DDM, 10 mM imidazole and protease inhibitors (Buffer A). After washing with 20 column volumes of buffer A containing 25 mM imidazole, the protein was eluted with 4 column volumes buffer A containing 300 mM imidazole. The sample was concentrated with an Amicon ultra centrifugal filter with a 30 kDa cut-off and injected into a buffer A (without imidazole) preequilibrated Superdex 200 HighLoad 16/60 gel filtration column. Presence of the complex was confirmed by SDS-PAGE, fractions were pooled and protease inhibitors added. Formation of the BamAP4P5-BamCDE proteoliposome sample required the equimolar combination of the detergent solubilized proteins under mild rotation for 1 h at 4°C. Prior to reconstitution into 1,2-dilauroyl-sn-glycero-3phosphocholine (DLPC; Avanti) liposomes, the appropriate amount lipids were dried for a lipid-to-protein molar ratio (LPR) of 50:1. The protein complex was then added and dialyzed at 4°C against 20 mM sodium phosphate pH 7 until liposomes were formed.

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Isotope labeled variants of the BamCDE and BamABCDE complexes in native OM were prepared as follows: For the BamABCDE complex E.coli BL21 star cells were transformed with the pJH114 (Roman-Hernandez et al., 2014) plasmid, for BamCDE transformation was carried out as described above. Minimum M9 medium cultures were inoculated and grown at 37°C to an optical density of ~1-1.2 after which they were placed at 25°C for 30 min before 0.5 mM IPTG was added. After an additional 25 min cells are spun down at 3 000xg for 10 min before being resuspended in fresh deuterated minimum M9 medium (99% D2O) containing

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deuterated BamCDE sample or 13

C,

15

15

C glucose and

N NH4Cl for the

13

C glucose and

13

C,

15

N, 2H-fractionally

15

N NH4Cl for the uniformly

N-labeled BamABCDE sample. Rifampicin was added to a final

concentration of 100 µg/ml in addition to 0.5 mM IPTG and cultures were grown overnight at 18°C. Cells were harvested by spinning at 4 000xg for 20 min and resuspended in 50 mM Tris-HCl pH 8.0, 2 mM EDTA, 25% sucrose and protease inhibitor. After cell lysis by French press, debris and large particulates were removed by multiple centrifugations at 4 000xg until the pellet is non-existent. The OM was harvested by ultracentrifugation at 22 000xg for 30 min, resuspended in 20 mM sodium phosphate pH 7 and ultracentrifuged at 90 000xg for 1h. (Beckman optima MAX-XP Ultracentrifuge with a TLA-55 rotor). NMR experiments and Data analysis Liposomes and OM preparations were filled into 3.2 mm or 1.3 mm magicangle spinning (MAS) rotors (Bruker Biospin) by ultracentrifugation (using a Beckman Coulter Optima L-90K ultracentrifuge with a SW32 Ti rotor) at 60 000xg for 1h at 4°C. (13C,13C) or

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N,1H experiments were subsequently

performed at static magnetic fields of 16.4, 18.8 and 22.3 Tesla, corresponding to 700, 800 and 950 MHz 13

1

H resonance frequencies

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respectively. ( C, C) spin diffusion experiments typically used 30 ms mixing times with PARIS (Weingarth et al., 2009) irradiation using MAS rates of 13 kHz. 1H detected NH experiments were performed at 60 kHz MAS on an 800 MHz system. For proton decoupling, SPINAL64 (Fung et al., 2000) was applied with 78 kHz irradiation on 1H at 13 kHz MAS. At 60 kHz MAS, low-

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power PISSARRO decoupling (~15 kHz irradiation) (Weingarth et al., 2008) was applied in both indirect and direct acquisition dimensions. Data were zero-filled and apodized using shifted squared sine-bell functions. Spectra were processed using Topspin (Bruker BioSpin). S.I. Table 1 summarizes the spectra recorded for all samples and their experimental details. For analysis of our spectra, we utilized previously reported solution-state NMR assignments for BamC (BMRB 16305) and BamE (BMRB 16926). In the case of BamD, for which no solution NMR assignments exist, we utilized ShiftX2 (B. Han et al., 2011) and the X-ray structure PDB 3TGO to estimate 1

H,

13

C and

15

N chemical shifts. All chemical-shift information was processed

using FANDAS (Gradmann et al., 2012) to compare predicted and experimentally determined ssNMR data sets. Analysis of the secondary structural conformations of various residues was performed essentially as described in (Etzkorn et al., 2008). Briefly, average CA-CB chemical shifts and the associated standard deviations (Seidel et al., 2009) of a specific amino acid in different secondary structures are plotted onto the ssNMR spectrum highlighting regions where specific amino acids, in typical secondary structures would give rise to correlations.

Results and Discussion Influence of environment and binding partner (BamA) on the BamCDE subcomplex To study the effect of the membrane environment and binding partner (BamA) on the structural arrangement of the 70 kDa subcomplex BamCDE, we utilized a combination of targeted labeling (Baker et al., 2015a) for in-situ OM preparations – Fig. 1F and 1G, and reconstitution into synthetic bilayers (Fig. 1E). We first evaluated the spectra of the BamCDE subcomplex in its natural environment in the absence of binding partner BamA (Fig. 2). The resulting spectrum showed a superposition of typical protein backbone and side-chain correlations that should exclusively stem from isotope-labeled BamCDE (see (Baker et al., 2015a)) as well as additional signal patterns that most likely stem from isotope-labeled lipids (indicated by the arrows in the

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(13C,13C) spectrum in Fig. 2A). As described by (Baker et al., 2015a), such signals could be further suppressed by 15N-edited correlation experiments.

Figure 2. ssNMR of native E. coli OM with over-expressed uniformly 13C, 15Nlabeled BamCDE A. 30 ms (13C, 13C) PARIS spin-diffusion experiment on uniformly labeled BamCDE in the outer membrane of E. coli. The lowest contour level is plotted at 3 x the estimated noise. Arrows point to correlations stemming from nonproteinaceous components present in the outer membrane. B. and C. Regions of interest which exhibit less correlations than expected, especially for CA-CB correlations – Serine and Threonine (B), Leucine, Aspartic acid and Asparagine (C). Crosses in panels B and C derive from solution NMR assignments for BamC (red) and BamE (orange) (BMRB 16035 (Knowles et al., 2009) and 16926 (Kim et al.,

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2011b)) and predicted backbone correlations for BamD (blue). Dashed boxes highlight regions of predominantly β-strand conformations for the corresponding amino acids.

For spectral analysis, we followed our earlier strategy (Pinto et al., n.d.; Sinnige et al., 2015a) whereby we make use of the existing solution NMR assignments for the soluble, isolated proteins, BamC (red, BMRB 16035 (Knowles et al., 2009)) and BamE (orange, BMRB 16926 (Kim et al., 2011b)). In the latter case multiple datasets/structures exist. However the calculated structure for BMRB 16926 (PDB 2KXX) has a lower backbone RMSD to the BamE structure from PDB 5D0Q. For BamC both solution-state NMR datasets are in excellent agreement, and also share a high similarity with the X-ray structure, with the exception of an extension of the helix present at the Cterminus of D1. This allows us to compare the overall similarity and fold of these proteins in the membrane-embedded complex. In addition, we generated a predicted (13C,13C) signal set in FANDAS for the mostly helical BamD protein based on the X-ray structure of the BamCD subcomplex (PDB 3TGO, (Kim et al., 2011a)indicated in blue, Fig. 2B and 2C). This approach highlights regions (including those indicated by the dashed boxes in Figures 2B and 2C) where one would expect CA-CB correlations to occur for serine and leucine/asparagine/aspartic acids predominantly in β-strand and random coil conformations. However, we observe that the experimental correlations are reduced in these areas when compared to, for example, the predominantly helical region for serine (58-63 ppm Fig. 2B). Our attempts to incorporate the BamCDE subcomplex into synthetic liposomes was unsuccessful due to degradation of BamC during dialysis (S.I. Fig. 1). This degradation could be prevented by addition of the unlabeled BamAP4P5 construct to the isotope labeled BamCDE subcomplex prior to reconstitution into liposomes (Fig. 3, green) and serves as the closest comparison to the BamCDE OM sample (Fig. 3, grey).

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Figure 3. Comparison of (13C, 13C) correlation spectra of BamCDE in E. coli outer membranes and in proteoliposomes with BamAP4P5. A. (13C, 13C) experiment of the uniformly 13C, 15N-labeled BamCDE complex in a membrane environment, with and without BamA (spectra in green and grey respectively). Panels B, C. and D. highlight regions of the spectrum in panel A where signal intensity increases upon formation of the BamAP4P5-BamCDE complex. Crosses in panels B, C and D derive from solution NMR assignments for BamC (red) and BamE (orange) (BMRB 16035 (Knowles et al., 2009) and 16926 (Kim et al., 2011b)) and predicted backbone correlations for BamD (blue). Dashed boxes highlight regions of the spectra mentioned in the text of predominantly β-strand, but also random coil conformations, for the amino acids mentioned.

Direct comparison of the (13C,13C) PARIS 30 ms mixing spectra obtained for the proteoliposome BamAP4P5-BamCDE and BamCDE in OM samples is not straightforward given their dissimilar environments. However, overall the spectra for the BamCDE OM preparation show less correlations in several

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spectral regions, including the CO-CA region of threonine, the CA-CB region of serine and CA-CB regions of leucine, aspartate, asparagine (Fig. 3 panels B-D respectively, regions in dashed boxes). Analysis of the experimental (BamC and BamE, Fig. 3 red and orange crosses) and predicted (BamD, Fig. 3 blue crosses) assignments in these regions, as well as distributions of these amino acids throughout the secondary structures of these proteins (S.I. Table 2), suggests that the increase observed in correlations for the proteoliposome sample occurs predominantly via stabilization of the folded domains of BamC.

Identification of BamC folded domains To gain further insight into the arrangement of BamC and other BAM subdomains in the assembly of the complex, we conducted 1H detected ssNMR experiments on fractionally deuterated BamCDE in complex with unlabeled BamA (Fig. 4). As in the previous section, we made use of solutionstate NMR 1H,

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N,

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CA assignments of the soluble BamE (Fig. 4A, orange)

and BamC (Fig. 4A, red) proteins and the predicted chemical shifts for backbone atoms of BamD (Fig. 4A, blue). Recently, we have shown the utility of such an approach for the case of P4 and P5 domains of BamA (Pinto et al., n.d.). In these studies, we compared P4 and P5 solution NMR assignments (Sinnige et al., 2015b) to results of 1H detected ssNMR spectra using proteoliposome BamAP4P5 preparations. Using 2D NH and 3D correlation experiments (CaNH, CoNH), we could account for at least 50% of P4P5 resonances that did not exhibit spectral overlap and agreed within 1 ppm in all spectral dimensions (1H,

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C,

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N) with the earlier solution-state NMR

assignments. Utilizing this approach for the analysis of the 2D NH spectrum (S.I. Fig. 2) shows, as expected, that the BamD protein predictions are indicative of a mostly helical conformation (see Fig. 4A) that mainly fall in the crowded region of this spectrum, between 7.5 and 9 ppm 1H. On the other hand, correlations typical for β-strand residues partly overlay with solution NMR assignments for soluble BamE (orange) and BamC (red).

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Figure 4. 1H detected 2D N-H correlation experiment on the proteoliposome preparation of the fractionally deuterated BamAP4P5-BamCDE complex. A. 2D NH spectra of the uniformly 13C, 15N-labeled, fractionally deuterated BamCDE complex with unlabeled BamA measured on an 800 MHz spectrometer. Predictions and assignments for BamCDE (in red, blue and orange, respectively) are added to the spectrum. B) Solution-NMR assignments for the BamC protein are colored in accordance with their localization within the domain organization of this protein (purple for the n-terminal tail and inter-domain linker), green for folded domain 1 (D1) and black for the folded domain 2 (D2) as in the cartoon.

Next, we examined in further detail whether these data would be consistent with the observation of both folded domains of BamC (Fig. 4B). For this purpose we color-coded assignments for residues 26-100 (extended random

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coil region that interacts extensively with BamD) and inter-domain linker in purple, domain 1 (101-222 in green) and domain 2 (229-344 in black). In comparison to Fig. 4A, which includes resonance (predictions) for BamD and BamE, hence suggests that both domain 1 (D1) and domain 2 (D2) are visible in our spectra. This notion was further supported by the analysis of a 3D CaNH spectrum (Fig. 5A) suggesting that those correlations that overlay well with previous solution-state NMR are found in BamC regions exposed to the solvent and thus distant from the interaction interface seen in X-ray structures. It is therefore not surprising that solution and ssNMR assignments are similar. (Fig. 5B, residues in cyan). Exceptions exist, namely E109, Q273 and S315 from BamC, which interestingly form part of the BamA-BamC interaction interface in the x-ray structure, and N249 that lies at the interaction surface between BamC D2 and BamD.

Figure 5. Solution-state NMR assignments for BamC and BamE which agree with ssNMR spectra. A. Strips of the 3D CANH spectrum that highlights the agreement of solution-state NMR assignments with ssNMR data for BamE (T39), BamC D1 (S122) and BamC D2 (S286). B. Residues for which the solution NMR assignments agree within 1 ppm in each frequency for the BamC (red) and BamE (orange) proteins are plotted onto the BamA-BamCDE structure as cyan sticks (PDB 5D0Q). BamA (purple) and BamD (blue) are represented as semi-transparent surfaces for simplicity.

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Due to the spectral crowding observed in the prior spectra, and in order to confirm that in fact the BamC domains are visible in our preparations, we recorded additional 2D (13C,13C) spin diffusion spectra (40 and 500 ms PDSD mixing) on the AVLMTS specifically labeled BamCDE in complex with unlabeled BamAP4P5 in liposomes. As shown before (Etzkorn et al., 2007; Sinnige et al., 2015a), such “forward” isotope labeling enhances the possibilities of extracting sequential correlations due to the reduction of spectral overlap. In Figure 6A, possible sequentials within the BamC domain with this labeling scheme are highlighted in bold. Figure 6B shows spectral cut-outs of short (40 ms, red) and long (500 ms, green) proton-driven spin diffusion experiments (see also ref. (Seidel et al., 2004)). Experiments recorded with these mixing times should yield short (40 ms) and long range (500 ms) correlations, with the latter one exhibiting sequential correlations that are characteristic and unique, in the context of the BamCDE complex, for D1 (Met201-Met202) and D2 (Leu283-Ser284) of BamC, respectively and match with previous (solution-state) resonance assignments. Taken together these results suggest that the presence of BamA is sufficient to induce stabilization of the folded domains D1 and D2 of BamC in synthetic and in natural membranes.

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Figure 6. Tentative identification of BamC folded domains. A. Topology and secondary structural elements of the BamC folded domains determined from the solution NMR assignments (BMRB 16035) using TALOS+ 24 are depicted above the sequence for these domains. In bold are the sequential AVLMTS residues specifically labeled in the BamAP4P5-BamCDE sample for the BamC protein. In red are sequentials identified in B. B. and C. Cut-outs of 40 (red) and 500 (green) ms PDSD spectra which highlight sequentials of residues unique to the BamC protein folded domains, L283-S284 and M201-M202 respectively. Black and blue crosses show intra- and inter-residue correlations, respectively, for the unique sequential pairs derived from the solution NMR chemical shifts of BamC (BMRB 16035).

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Global overview of the 200 kDa BamABCDE complex in native environment Finally, in order to assess the overall fold of the 200 kDa complex, uniformly 13

C,

15

N-labeled BamABCDE in E. coli OM was produced utilizing the

aforementioned approach that exploits inhibition of endogenous E. coli gene expression via rifampicin (Baker et al., 2015a). Overall, the (13C,13C) correlation spectrum recorded on a 950 MHz spectrometer with 30 ms mixing suggests that the BamABCDE proteins are structured in these cellular preparations (Fig. 7). Isolated amino-acid regions, such as those shown in Fig. 7B for serine and threonine, and Fig. 7C for alanine, also highlight the distribution

of

these

residues

throughout

all

secondary

structural

conformations. In particular, we observe strong correlations reflecting αhelical Alanine residues which are most abundant in BamD (S.I. Table 2).

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Figure 7. ssNMR on native E. coli outer membranes with over-expressed BamABCDE. A. 30 ms (13C, 13C) correlation spectrum of uniformly 13C, 15Nlabeled BamABCDE, 200kDa complex, in E. coli outer membrane. The lowest contour level is plotted at 4x the estimated noise. Boxes highlight regions of the spectrum that exhibit dispersion consistent with amino acids in the various forms of secondary structure. B and C. Zoom-in of serine/threonine and alanine regions respectively with dashed boxes to indicate average chemical shift values (CSI) for the indicated secondary structures (α - α-helix; β - β-strand and r.c. – random coil). Colors represent the Bam complex components as in the cartoon in panel A.

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Conclusions
 A detailed analysis of membrane protein assembly is critical for understanding membrane protein function, including signal transduction and amplification (Kaplan et al., 2016b; Maciejko et al., 2015), or as is the case of BAM, for substrate recognition and membrane insertion. Such a function requires not only the correct assembly of the complex but is also intimately tied to the environment wherein the complex finds itself – the bacterial OM. Previous structural studies of the complex have employed membrane mimetics. Here we have shown how cellular ssNMR in combination with ssNMR data on synthetic liposomes preparations and solution-state NMR results on soluble BAM constructs can be used to infer atomic-level insight into the fold of the BAM complex as well as of BAM subcomplexes in the OM. Our studies suggest that the BamCDE subcomplex exhibits increased structural or dynamical disorder in BamC and possibly also in the BamE protein. In the case of BamC, these findings are in agreement with the observed proteolytic sensitivity of BamC observed previously in detergent (Webb et al., 2012) and support the hypothesis of a dynamic protein that exposes protease sensitive sites in the absence of a binding partner. Addition of the BamAP4P5 construct leads to a significant structural stabilization of BamC, as evidenced by 1H detected ssNMR experiments on uniformly

13

15

13

N-labeled, fractionally deuterated BamCDE as well as by (13C,

C,

C)

correlation experiments using forward-labeled BamCDE in complex with unlabeled BamAP4P5. This stabilization includes the domain 2 of BamC. Interestingly, the recently published structures for the full BamABCDE complex observed two states for the BamA protein, the central component of the complex. A striking similarity between these structures refers to the absence of X-ray density (and later EM density) for the C-terminal folded domain 2 of BamC despite the absence of degradation of this protein in the crystals. This domain only appears in one molecule of the asymmetric unit of one of the four obtained structures (PDB 5D0Q), an aspect that has been attributed to transient crystal packing based on molecular dynamics

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simulations (Gu et al., 2016). Our data suggest that this domain is stabilized by the presence of the BamAP4P5 construct, an observation that is striking given that in the X-ray structures, this transient BamA-BamC interaction occurs via POTRA 2, a domain we lack in our samples. Whilst domain 1 of BamC is stabilized through its interaction with BamD, the second domain is highly flexible in the case without BamA, an observation that holds true even when the complex, BamCDE, is located in its native environment. Further studies will be needed to determine whether these domains reside in the periplasmic space along with BamD and BamE or if they are located on the extracellular surface of the OM (Webb et al., 2012). Finally, we have presented ssNMR data on the entire BAM complex in native membranes that suggest that, on the global level, all subdomains adopt the general folds observed for their detergent solubilized counterparts. A more detailed analysis should be possible by using advanced isotope-labelling methods that allow us to zoom in into the specific regions of the complex, for example before or during substrate interactions.

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Acknowledgments The authors would like to thank Daniel Kahne for kindly providing the pSK46 and pBamE-His plasmids and Harris Bernstein for providing the pJH114 plasmid. We would also like to thank Klaartje Houben for her help with setting up ssNMR experiments on the 950 MHz instrument. This work was funded in part by Netherlands Organization for Scientific Research (NWO) (grants 700.26.121 and 700.10.443 to M.B,) and iNEXT (project number 653706), a Horizon 2020 program of the European Experiments at the 950 MHz instrument were supported by uNMR-NL, an NWO-funded National Roadmap Large-Scale Facility of the Netherlands (grant number: 184.032.207). References Andreas, L.B., Le Marchand, T., Jaudzems, K., Pintacuda, G., 2015. Highresolution proton-detected NMR of proteins at very fast MAS. Journal of Magnetic Resonance 253, 36–49. Asami, S., Reif, B., 2013. Proton-detected solid-state NMR spectroscopy at aliphatic sites: application to crystalline systems. Acc. Chem. Res. 46, 2089–2097. doi:10.1021/ar400063y Bakelar, J., Buchanan, S.K., Noinaj, N., 2016. The structure of the β-barrel assembly machinery complex. Science 351, 180–186. doi:10.1126/science.aad3460 Baker, L., Sinnige, T., Schellenberger, P., de Keyzer, J., Siebert, C.A., Driessen, A.J.M., Baldus, M., Grunewald, K., 2017. Native Membrane Structural Biology: Combining proton-detected solid-state NMR and electron cryotomography to study membrane proteins across resolutions in their native environment. Structure in press. Baker, L.A., Baldus, M., 2014. Characterization of membrane protein function by solid-state NMR spectroscopy. Curr. Opin. Struct. Biol. 27, 48–55. doi:10.1016/j.sbi.2014.03.009 Baker, L.A., Daniëls, M., van der Cruijsen, E.A.W., Folkers, G.E., Baldus, M., 2015a. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling. J. Biomol. NMR 62, 199–208. doi:10.1007/s10858-015-9936-5 Baker, L.A., Folkers, G.E., Sinnige, T., Houben, K., Kaplan, M., van der Cruijsen, E.A.W., Baldus, M., 2015b. Magic-Angle-Spinning Solid-State NMR of Membrane Proteins, 1st ed, Ion Channels Part B. Elsevier Inc. doi:10.1016/bs.mie.2014.12.023 Bos, M.P., Robert, V., Tommassen, J., 2007. Biogenesis of the GramNegative Bacterial Outer Membrane. Annual Review of Microbiology 61, 191–214. doi:10.1146/annurev.micro.61.080706.093245 Etzkorn, M., Kneuper, H., Dünnwald, P., Vijayan, V., Krämer, J., Griesinger, C., Becker, S., Unden, G., Baldus, M., 2008. Plasticity of the PAS domain

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S.I. Figures and Tables

S.I. Figure 1. BamCDE sample preparations. A. Outer membrane preparation of BamCDE prepared utilizing the rifampicin approach. Note the high abundance of OMP and the very low contribution of additional OMPs. B. Purified BamCDE after gel filtration (1) and after a few days of dialysis (2).

S.I. Table 1 Solid-state NMR data collection conditions for the various samples and their labelling schemes. BamCDE Labelling Environment Experiment

Spectrometer (MHz) MAS (kHz) Experimental time Temperature (kelvin)

BamAP4P5-BamCDE 13 15 C, N C, N AVLMTS

13

15

BamABCDE

E. coli OM

DLPC LPR 50:1

DLPC LPR 50:1

PARIS 30ms

PARIS 30 ms

PDSD 40 and 500 ms

C, N, 2 H DLPC LPR 50:1 NH and CaNH

700

700

950

800

950

13

13

13

60

13

9.9d

1.8d

1.4d (40 ms) 4.5d (500 ms)

5.5d

1.6d

262

262

255

298

255

13

15

13

C, N

15

13

15

C, N

E. coli OM PARIS 30 ms

S.I. Table 2. Distribution of amino acid residues within secondary structural elements in the BamCDE complex. Secondary structural conformations were determined for BamC (BMRB 16305) and BamE (BMRB 16926) using TALOS+ on the corresponding solution-state NMR datasets. For BamD the secondary structures are those as determined by the x-ray structure PDB 5D0O.

BamC Coil

Alanine Arginine

17 8

BamD

Helix Strand Coil

16 3

5 4

2 2

BamE

Helix Strand Coil

25 14

0 0

1 4

Helix Strand

3 0

0 0

27

Asparagine Aspartic acid Glutamine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

17 6 9 6 21 2 4 13 7 3 0 14 12 17 0 5 8

S.I. Figure 2.

1

6 4 8 3 1 0 1 7 2 3 3 2 9 2 3 1 8

2 3 8 1 2 0 2 9 4 2 2 0 5 3 2 4 10

5 6 2 0 3 1 0 0 1 1 0 3 7 3 0 3 2

9 12 16 8 6 1 8 18 8 6 7 9 4 8 2 13 10

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

5 2 4 2 6 1 2 3 0 1 1 3 3 5 0 2 4

1 1 3 0 2 0 0 2 1 1 1 1 3 0 0 1 3

2 1 1 0 0 0 1 3 1 0 2 1 0 6 1 1 1

H detected 2D N-H correlation experiment on the proteoliposome preparation of the fractionally deuterated BamAP4P5-BamCDE complex. 2D NH spectra of the uniformly 13C, 15N-labeled, fractionally deuterated BamCDE complex with unlabeled BamA measured on an 800 MHz spectrometer.

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