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. Author manuscript; available in PMC: 2015 Aug 5.
Published in final edited form as: Structure. 2014 Jul 10;22(8):1204–1209. doi: 10.1016/j.str.2014.05.016

NMR Polypeptide Backbone Conformation of the E. coli Outer Membrane Protein W

Reto Horst 1,3, Pawel Stanczak 1,4, Kurt Wüthrich 1,2,*
PMCID: PMC4150354  NIHMSID: NIHMS614091  PMID: 25017731

SUMMARY

The outer membrane proteins (Omp) are key factors for bacterial survival and virulence. Among the Omps which have been structurally characterized either by X-ray crystallography or by NMR in solution, the crystal structure of OmpW stands out because three of its four extracellular loops are well defined, whereas long extracellular loops in other E. coli Omps are disordered in the crystals as well as in NMR structures. OmpW thus presented an opportunity for detailed comparison of the extracellular loops in a β-barrel membrane protein structure in crystals and in non-crystalline milieus. Here the polypeptide backbone conformation of OmpW in 30-Fos micelles was determined. Complete backbone NMR assignments were obtained and the loops were structurally characterized. In combination with the OmpW crystal structure, NMR line shape analyses and 15N{1H}-NOE data, these results showed that intact regular secondary structures in the loops undergo slow hinge motions at the detergent–solvent interface.

INTRODUCTION

Outer membrane proteins (Omp) are abundant integral membrane proteins (IMPs) in E. coli. They have important roles for bacterial survival and virulence (Smith et al., 2007; Weiser and Gotschlich, 1991), and in E. coli infections they mediate key processes such as cell-adhesion (Torres and Kaper, 2003), host invasion (Prasadarao et al., 1996), and immune evasion (Prasadarao et al., 2002; Weiser and Gotschlich, 1991). Due to their important physiological roles, E. coli Omps have been extensively investigated, and several three-dimensional structures have been determined by X-ray crystallography (Hong et al., 2006; Pautsch and Schulz, 1998; Vogt and Schulz, 1999), and by NMR in solution (Arora et al., 2001; Edrington et al., 2011; Fernandez et al., 2001, 2004; Hagn et al., 2013; Hiller et al., 2008; Hwang et al., 2002; Liang and Tamm, 2007).

Structurally, the E. coli outer membrane proteins form transmembrane β-barrels carrying extracellular loops of variable lengths. Complete structures of OmpX, which contains only short loops, were determined both by X-ray crystallography (Vogt and Schulz, 1999) and by NMR in solutions of detergent micelles (Fernandez et al., 2004) and nanodiscs (Hagn et al., 2013). Only incomplete structure determinations were obtained for outer membrane proteins which contain one or several long extracellular loops, because of structural disorder in the crystals (Pautsch and Schulz, 1998, 2000) and deterioration of the NMR spectra due to slow conformational exchange processes (Arora et al., 2001; Fernandez et al., 2001; Liang and Tamm, 2007). OmpW has been an exception, since three of its four long extracellular loops are well structured in crystals, mainly due to favorable intermolecular contacts, which include a well-ordered molecule of the detergent LDAO in the loop region (Hong et al., 2006). This paper presents a NMR determination of the polypeptide backbone conformation in OmpW, where complete backbone NMR assignments enabled a detailed characterization of dynamic conformational plasticity in the extracellular parts of the molecule. The NMR structure determination thus provided a basis for a detailed description of OmpW in an environment that mimics its milieu on the cell surface in contact with aqueous body fluids. This new information was evaluated with regard to improved understanding of structure–function correlations in E. coli outer membrane proteins and β-barrel integral membrane proteins from other organisms.

RESULTS

Previous work with E. coli outer membrane proteins showed that the choice of the detergent and the protein-to-detergent molar ratio are critical factors for obtaining high-quality multidimensional NMR spectra (Stanczak et al., 2009; Zhang et al., 2008). For OmpW, we performed an extensive micro-coil NMR screen in search of solution conditions that would yield “structure-quality” NMR samples (Stanczak et al., 2012). As a result, we used for the present structure determination a solution of 1.2 mM uniformly [2H,13C,15N]-labeled OmpW reconstituted in 230 mM 30-Fos (2-undecylphosphocholine) containing 5 mM phosphate buffer at pH 6.8, 10 mM NaCl and 0.3% (v/w) NaN3. All data were recorded at 308 K.

Complete NMR Assignments were obtained for the OmpW Polypeptide Backbone

Based on the connection pathways provided by six TROSY-type triple resonance experiments (Figure S1; for details see Experimental Procedures), sequence-specific resonance assignments for 95% of the protein backbone were obtained. This contrasts with previous NMR studies with the E. coli outer membrane proteins OmpA (Arora et al., 2001; Fernandez et al., 2001) and OmpG (Liang and Tamm, 2007), where parts of the long extracellular loops could not be assigned. In OmpW, the residual unassigned residues, i.e. Q39, K65, F110, V139, Y157, M158, D159, L179, D180 and F184, were not clustered in continuous segments of the polypeptide chain, but most of them appear in polypeptide segments that connect the transmembrane barrel with the extracellular loop-region (Figures 1 and 2). These missing assignments did not prevent to unambiguously establish connections between the transmembrane β-barrel and the extracellular loops. As is discussed in detail below, the absence of these assignments is due to line broadening beyond detection by conformational exchange.

Figure 1. Survey of NMR Assignments obtained for OmpW in 30-Fos Micelles.

Figure 1

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(a) Graphical presentation of the sequence-specific polypeptide backbone NMR assignments obtained from TROSY-type triple resonance experiments. For each residue the 1HN, 15N, 13Cα, 13Cβ and 13CO chemical shift assignments are indicated by vertical bars in the respective rows. Sequence positions are indicated at the top of each panel. (b) Locations in OmpW of residues without assigned 15N–1H correlation NMR signals (magenta) and residues with weak cross-peak intensities in 2D [15N,1H]-TROSY spectra (cyan). The β-strands forming the trans-membrane β-barrel are indicated.

Figure 2. Signal intensities in the 2D [15N,1H]-TROSY correlation spectrum and 15N{1H}-NOEs in OmpW reconstituted in 30-Fos micelles.

Figure 2

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(a) Plot of the 2D [15N,1H]-TROSY cross-peak signal intensities versus the amino acid sequence. Residues which showed either line broadening or evidence for line doubling (see text) are highlighted in cyan. (b) Plot of 15N{1H}-NOE intensities versus the sequence. Values between 0.5 and 1.0 indicate well-structured parts of the protein; values < 0.5 manifest increased flexibility on the sub-nanosecond timescale. The positions of the regular secondary structures are indicated at the top, with the β-strands forming the trans-membrane β-barrel indicated by asterisks.

Molecular Architecture of OmpW in 30-Fos Micelles

Sequence locations of regular secondary structures were determined interactively from analysis of 13C chemical shifts, and from a structure calculation with the software CYANA (Güntert et al., 1997). For the interactive approach, differences between 13Cα and 13Cβ chemical shifts observed in OmpW and the corresponding random coil shifts, ΔCα and ΔCβ, were evaluated to obtain values of (ΔCα −ΔCβ) (Figure S2). Segments with continuous trains of large negative or positive (ΔCα −ΔCβ) values are indicative of β-strands and helical structures, respectively (Metzler et al., 1993). On this basis, the patterns in the plot of ΔCα −ΔCβ versus the OmpW sequence (Figure S2) match the sequence locations of the 13 β-strands and the single helix in the crystal structure of OmpW (Hong et al., 2006; PDB ID: 2F1V), if one allows for the missing assignments of the three-residue segment 157–159 in the trans-membrane barrel part of β11 (Figure 1).

For the structure calculation with the program CYANA (Güntert et al., 1997), the initial input included 224 upper distance limits derived from 1H–1H NOEs, and 240 dihedral angle constraints derived from the 13C chemical shifts, as described in Experimental Procedures (Table 1). In the ensemble of conformers resulting from the converged CYANA calculations, it was readily apparent that a global superposition of the entire polypeptide chain for best fit with the mean coordinates would not provide a satisfactory result. However, the β-strands in Figure S2 which coincide with the sequence locations of the transmembrane β-barrel could be superimposed with a rmsd value of 2.0 Ǻ relative to the mean coordinates, affording a clear view of the β-barrel. Using the DSSP algorithm (Kabsch and Sander, 1983), 68 hydrogen bonds were identified in this preliminary NMR structure. After addition of the hydrogen bond constraints to the input for the CYANA structure calculation, the polypeptide backbone fold visualized in Figure 3, a–d, was obtained.

Table 1.

NMR structure calculation for [2H,13C,15N]-OmpW in 30-Fos micelles at 308Ka

Quantity Value
NOE upper distance constraintsa 224
Dihedral angle constraintsa 240
Hydrogen bond constraintsb 68
Residual target function value (Å2) 9.16 ± 3.0a
Residual NOE violations
  Number > 0.1 Å 3
  Maximum (Å) 1.3
Residual dihedral angle violations > 2.0 deg 0
Average backbone rmsd valuesc
  Polypeptide segments a (Fig. 4) 1.37
  Polypeptide segments b (Fig.4) 1.18
  Polypeptide segment c (Fig.4) 0.67
  Polypeptide segments d (Fig.4) 1.78
Ramachandran plot statisticsd (%)
Most favored regions 80.7
Additional allowed regions 18.0
Generously allowed regions 1.3
Disallowed regions 0.0
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a

Details of the data collection and the structure calculation with the software CYANA (Güntert et al., 1997) are described in Experimental Procedures.

b

Hydrogen bond constraints were added with the DSSP algorithm (Kabsch and Sander, 1983) applied to a structure calculated from the NOE upper distance constraints and the dihedral angle constraints alone.

c

Rmsd values relative to the mean coordinates were calculated for the backbone atoms N, Cα and C’ of the following polypeptide segments, which are visualized in Figure 4. a: 7–17, 37–47, 52–59, 83–88, 98–107, 132–143, 150–155 and 183–191 (corresponds to the trans-membrane β-barrel). b: 125–129, 162–167 and 173–177 (corresponds to the extracellular β-sheet with the strands β9, β11 and β12). c: 114–124 (corresponds to α1). d: 33–34, 63–68, 73–80 (corresponds to the extracellular β-sheet with the strands β2, β5 and β6).

d

The Ramachandran plot statistics were calculated using the PSVS software suite (psvs-1_5-dev.nesg.org) with the following residues that were classified as ordered by PSVS: 5–11,13–16,42–47,52–59,82–88,105–109,116–123,141–144,148–156,160–164,167–169,172–178 and 183–188.

Figure 3. NMR Polypeptide Backbone Structure of OmpW in 30-Fos Micelles and Comparison with the Crystal Structure.

Figure 3

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Local superpositions for best fit to the mean atom coordinates of the 20 CYANA conformers with the lowest residual target function values are shown for the following polypeptide segments (Table 1): (a) 7–17, 37–47, 52–59, 83–88, 98–107, 132–143, 150–155 and 183–191 (corresponds to the trans-membrane β-barrel, highlighted in cyan). (b) 125–129, 162–167 and 173–177 (corresponds to the extracellular β-sheet with the strands β9, β11 and β12, highlighted in magenta). (c) 114–124 (corresponds to α1, highlighted in green). (d) 33–34, 63–68, 73–80 (corresponds to the extracellular β-sheet with the strands β2, β5 and β6, highlighted in yellow). (e) X-ray structure of OmpW reconstituted with LDAO (PDB id: 2F1V) shown as a ribbon diagram. The color-code matches the colors of the parts of the NMR structure in (a) to (d). In the center, the thickness of the lipophilic phase of the E. coli bilayer outer membrane is indicated.

In the final result of the structure calculation, a bundle of 20 conformers selected for the lowest residual target function values could be locally superimposed for best fit of the transmembrane β-barrel with a RMSD of 1.37 Ǻ (Table 1, Figure 3a; see also Figure S3). The height of the β-barrel matches the thickness of the lipophilic phase of the E. coli outer membrane of 27 Ǻ (Wimley, 2002). Both size and shape of the β-barrel agree closely with the crystal structure of OmpW in LDAO (Hong et al., 2006), as documented by the RMSD value for the structure superposition (Figure S3a). For the further characterization of the NMR structure, we generated local superpositions of the bundle of conformers for the regular secondary structures outside of the β-barrel. This approach revealed that the regular secondary structures in the long extracellular loops are locally well defined also for OmpW in 30-Fos micelles (Figure 3, b–d) and coincide closely with the crystal structure (Figure S3). This is due to a quite dense network of experimental constraints in the loops. For example, the two antiparallel β-sheets are defined by 14 and 6 long-range inter-strand NOEs, respectively.

Dynamics in the OmpW Backbone Conformation

The observations visualized in Figure 3, a–d, are compatible with the assumption that there are hinge motions of the extracellular loops which leave the regular secondary structures intact, with the hinges located near the surface of the β-barrel. To further characterize the implicated intramolecular motions, we analyzed the signal intensities and line-shapes of the cross-peaks in 2D [15N,1H]-TROSY correlation spectra and collected 15N{1H}-NOE data for OmpW in 30-Fos micelles.

Weak 15N–1H cross-peaks were found to be clustered in polypeptide segments that connect the transmembrane barrel with the extracellular loops, often next to residues without assigned signals (Figures 1 and 2a). For some residues the reduced peak intensity is due to line broadening, but for other residues we only observed loss of peak height (Figure S4, a–d). There is evidence for peak doubling, which would explain the reduced peak height for the latter group of residues. The additional peaks have low intensity and appear mostly as shoulders of stronger peaks (Figure S4e), and they could therefore not be individually assigned. Conformational exchange on the millisecond time scale can explain both phenomena, as well as line broadening beyond detection for the signals of residues without assigned resonances, since different exchange effects at a given rate can arise from variation among individual residues of the chemical shift differences between the exchanging structures. Additional measurements showed that with the exception of a single residue, all observed and assigned NMR signals have 15N{1H}-NOE values between 0.5 and 1.0 (Figure 2b), which is indicative of long-lived structures. Overall, the combination of peak shape analysis and measurement of heteronuclear NOEs complements the structural data in Figure 3, a–d, with timescales of the implicated hinge motions of the loops. While the extra-cellular loops of OmpW are involved in slow rate processes, on the millisecond timescale, the regular secondary structures in the loops are long-lived on the nanosecond timescale, which explains that they stay intact during the hinge motions (Figure 3).

DISCUSSION

The present determination of the dynamic polypeptide backbone architecture of the integral membrane protein OmpW from E. coli solubilized in 30-Fos micelles is based on complete sequence-specific backbone NMR assignments. Combined with information from the crystal structure (Hong et al., 2006), which includes a precise description of three of the four long extracellular loops, this enabled a detailed structural characterization of these loops in solution, in addition to describing the transmembrane β-barrel. Specifically, the NMR data show that the long extracellular loops are involved in intramolecular rate processes on the millisecond to microsecond timescale. This coincides with the results of earlier studies of E. coli Omps, where broadening of the NMR signals of residues linking long loops with the β-barrel led to the conclusion that there were slow rate processes (Arora et al., 2001; Fernandez et al., 2001; Liang and Tamm, 2007; Zhuang et al., 2003). The observations in OmpW provide a quite precise description of these dynamic processes, with indication of large-amplitude hinge motions of intact regular secondary structures in the long loops, with the hinges located at the β-barrel–loop interface. The rate processes are thus seen to involve major rearrangements of polypeptide backbone segments, so that the slow rate of the conformational exchange can readily be rationalized (Shaw et al., 2010).

The complete structural characterization of the polypeptide backbone architecture in OmpW is primarily a result of the extensive search for a NMR-friendly combination of the detergent used for the reconstitution, the solution conditions and the temperature (Stanczak et al., 2012). Previous reports on structural studies of OmpW include mention of failed attempts at reconstitution with various detergents (Hong et at., 2006), and we experienced similar failures at the outset of the studies with OmpW (Stanczak et al., 2012; unpublished results). Independently, investigations with different β-barrel proteins (Horst et al., 2012; Stanczak et al., 2009; Zhang et al., 2008) and with other classes of membrane proteins such as GPCRs (Horst et al., 2013) had demonstrated the critical importance of optional conditioning of integral membrane protein reconstitution in structural biology projects with solution NMR spectroscopy.

OmpW has been suggested to function as a transport or channel protein (Hong et al., 2006). This information appears so far to be rather hypothetical, and therefore we forego attempts at establishing specific correlations of the observations on the OmpW structure at the interface of the membrane surface and the surrounding aqueous body fluids (Figure 3) with the physiological function of OmpW. On a more general level, considering that most of the putative functions attributed to β-barrel outer membrane proteins involve interactions with other proteins or small ligand molecules (Fairman et al., 2011), it is interesting that these reaction partners can target intact regular secondary structures. Although these structures undergo the aforementioned hinge motions relative to the transmembrane β-barrel, they might be fixed in unique orientations relative to the β-barrel surface by the complexation with these reaction partners. In this context, it is worth pointing out that the implicated frequencies of the hinge motions (Figure 3) coincide with the frequencies of Phe and Tyr “ring flips” (Wüthrich and Wagner, 1975), which represent ubiquitous low-frequency, large-amplitude fluctuation modes in globular proteins (Shaw et al, 2010), so that there would be matching frequencies of conformational fluctuations in interactions of partner proteins with the extracellular surface of β-barrel outer membrane proteins.

EXPERIMENTAL PROCEDURES

We previously reported on micro-scale NMR screening of different detergents over a wide range of solution conditions for optimizing the reconstitution of OmpW (Stanczak et al., 2012) Here, milligram quantities of uniformly 2H,13C,15N-labeled OmpW were prepared for the structure determination and reconstituted with the detergent 30-Fos in the H2O solution described at the outset of the Results section. 30-Fos was obtained from Dr. Qinghai Zhang (Zhang et al., 2008).

NMR experiments

3D [15N,1H]-TROSY-HNCO, 3D [15N,1H]-TROSY-HN(CA)CO and 3D [15N,1H]-TROSY-HN( CO)CA experiments were recorded on an AVANCE-600 spectrometer (Bruker Billerica, MA), using a QCI 1H/19F–13C/15N quadruple-resonance cryoprobe with shielded z-gradient coil. The other experiments were recorded on an AVANCE-800 spectrometer, using a TXI 1H–13C/15N triple-resonance room temperature probe with shielded triple-axis gradient coils. The temperature was monitored with a standard sample of 4% methanol in methanol-d4. The following parameters were used for the individual experiments: 2D [15N,1H]-TROSY (Pervushin et al., 1997), data size 128(t1) × 1024(t2) complex points, t1max = 45 ms, t2max = 71 ms; 3D [15N,1H]-TROSY-HNCA (Salzmann et al., 1999b), 1024(t1) × 35(t2) × 30(t3) complex points, t1max = 14 ms, t2max = 8 ms, t3max = 71 ms; 3D [13C]-constant-time [15N,1H]-TROSY-HNCA (Salzmann et al., 1999a), 1024(t1) × 64(t2) × 32(t3) complex points, t1max = 12 ms, t2max = 10 ms, t3max = 71 ms; 3D [15N,1H]-TROSY-HN(CO)CA (Salzmann et al., 1999b), 1024(t1) × 35(t2) × 34(t3) complex points, t1max = 106 ms, t2max = 12 ms, t3max = 15 ms; 3D [15N,1H]-TROSY-HN( CA)CO (Salzmann et al., 1999b), 1024(t1) × 35(t2) × 34(t3) complex points, t1max = 106 ms, t2max = 8 ms, t3max = 15 ms; 3D [15N,1H]-TROSY-HNCO (Salzmann et al., 1998), 1024(t1) × 32(t2) × 30(t3) complex points, t1max = 106 ms, t2max = 11 ms, t3max = 14 ms; 3D [15N,1H]-TROSY-HNCACB (Salzmann et al., 1999b), 1024(t1) × 36(t2) × 36(t3) complex points, t1max = 1024 ms, t2max = 36 ms, t3max = 36 ms; 3D [1H,1H]-NOESY-[15N,1H]-TROSY (Pervushin et al., 2000), 1024(t1) × 40(t2) × 150(t3) complex points, t1max = 71 ms, t2max = 14 ms, t3max = 10 ms, mixing time τm = 200 ms. To suppress the signals from the detergent and the water, we replaced the standard WATERGATE element with REBURP(1H) pulses (Hilty et al., 2002) of duration 1.5 and 2.0 ms, respectively, at 1H frequencies of 800 MHz and 600 MHz. The carrier frequency offset was 8.5 ppm, and two sine-shaped pulsed field gradients were used, with a maximum gradient strength of 35 G/cm and a duration of 0.8 ms. For the triple resonance experiments, the ST2-PT element (Pervushin et al., 1998) was concatenated into the 15N-constant time period (Salzmann et al., 1999c).

Heteronuclear NOEs were collected using a 15N{1H}-NOE-TROSY experiment (Zhu et al., 2000), with the following acquisition parameters: data size 128(t1) × 1024(t2) complex points, t1max = 45 ms, t2max = 71 ms. The proton presaturation period was 3 s.

Structure Determination

The 3D [1H,1H]-NOESY-[15N,1H]-TROSY spectrum was interactively peak-picked, assigned and integrated with the software XEASY (Bartels et al., 1995), resulting in 224 upper distance constraints (Table 1). Dihedral angle constraints were determined from the 13Cα and 13Cβ chemical shifts, using the program TALOS (Cornilescu et al., 1999). Structure calculations were performed with the software CYANA 2.1 (Güntert et al., 1997). An initial calculation based on the NOE upper distance constraints and the dihedral angle constraints (Table 1) converged with a residual target function value of 3.8 Å2. Hydrogen bond constraints were then added to this initial structure using the DSSP algorithm (Kabsch and Sander, 1983), and a new round of structure calculations was initiated to generate 100 conformers. From this ensemble of converged structure calculations, we selected a bundle of 20 conformers on the basis of low residual target function values for the presentation of the NMR polypeptide backbone structure (Figure 3). All figures showing structural features of OmpW were prepared with MOLMOL (Koradi et al., 1996).

Supplementary Material

01

  • Structure and dynamics of OmpW solubilized in 30-Fos micelles have been determined

  • Regular secondary structures formed by extracellular loops undergo hinge motions

ACKNOWLEDGEMENTS

This work was supported by the NIH Roadmap initiative grant P50 GM073197 for technology development. Kurt Wüthrich is the Cecil H. and Ida M. Green Professor of Structural Biology at the Scripps Research Institute. We thank Dr. N. S. Bhavesh and Dr. F. F. Damberger for providing us with results from exploratory work toward reconstitution of OmpW, which was pursued in the laboratory of K.W. at the ETH Zürich in Switzerland, and Dr. G. Wagner for helpful discussions about the interpretation of the structural data on OmpW.

Footnotes

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ACCESSION NUMBERS

The chemical shift assignments have been deposited in the BMRB under accession code 19,637, and the atom coordinates for the polypeptide segments used for the superpositions in the four bundles of Figure 3, a–d, are in the PDB under accession code 2mhl.

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