Structure of the outer membrane porin OmpW from the pervasive pathogen Klebsiella pneumoniae

The crystal structure of the outer membrane protein OmpW from Klebsiella pneumoniae is reported.


Introduction
Outer membrane porins (OMPs) are an important class of �-barrel proteins that form water-filled channels in Gramnegative bacteria.They enable the diffusion of nutrients and the efflux of toxins across the outer membrane (Lou et al., 2009).From a clinical perspective, OMPs are important in modulating the diffusion of antibiotics into the bacterial cell, where mutations or reduced expression of the OMPs enhance antibiotic resistance (Page `s et al., 2008).It has also been shown that OMPs participate in F-like plasmid conjugation, a form of horizontal gene transfer where plasmids are transferred from donor to recipient bacteria in a contact-dependent manner (Lederberg & Tatum, 1946;Frankel et al., 2023).We have recently shown that the efficient conjugation of the multidrugresistant R100-1 plasmid into both Escherichia coli (EC) and Klebsiella pneumoniae (KP) relies on the formation of matingpair stabilization via interaction between the R100-1-encoded OM protein TraN� in the donor and the OMP OmpW EC or OmpW KP in the recipient (Low et al., 2022(Low et al., , 2023)).Pairing of the TraN isoform with recipient receptors mediates conjugation species specificity and host range; an in-depth review of mating-pair stabilization and the role of TraN has been provided by Frankel et al. (2023).In brief, TraN is an outer membrane protein that is composed of two domains, a base and an extended tip; the base consists of a conserved amphipathic �-helix that possibly anchors TraN to the OM, whereas the tip is mostly comprised of �-sheets linked to a �-sandwich domain.The loops at the tip function as a TraN sensor that participates in recipient selection (Frankel et al., 2023) In addition to its role in conjugation, OmpW contributes to virulence as the upregulation of OmpW EC increases resistance to host immune defence (Wu et al., 2013).Conversely, OmpW is a key antigen; indeed, OmpW-immunized mice show greater protection against bacterial infections.This could pave the way for the use of OmpW in vaccine preparation (Huang et al., 2015).
The crystal structure of OmpW EC forms an eight-stranded monomeric �-barrel with an extracellular region that is involved in hydrophobic substrate binding (Hong et al., 2006).Here, we present the crystal structure of OmpW KP at 3.2 A resolution and draw structural comparisons with OmpW EC , both of which are conjugation receptors for TraN�.

Macromolecule production
The mature protein sequence of OmpW KP (His22-Phe212) was subcloned into the pTAMANHISTEV vector in-frame with a tamA signal sequence followed by an N-terminal His 7 tag and a Tobacco etch virus (TEV) cleavage site, using the NcoI and XhoI restriction-enzyme sites.The construct was transformed into & Walker, 1996) and expressed in Terrific Broth (TB) medium.Cultures were incubated at 37 � C with orbital shaking at 200 rev min À 1 until an optical density at 600 nm (OD 600 ) of 0.6-0.8 was achieved.Cultures were then induced with isopropyl �-d-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM and maintained for 3 h.The cells were harvested by centrifugation (Beckman Coulter) at 8000g for 10 min and stored at À 80 � C. Outer membranes were prepared as described previously (Beis et al., 2006) and were then solubilized in phosphatebuffered saline (PBS) supplemented with 1% N,N-dimethyl-ndodecylamine N-oxide (LDAO) overnight.Unsolubilized membranes and debris were removed by ultracentrifugation at 131 000g for 1 h.The supernatant was supplemented with 30 mM imidazole and passed through a 5 ml HisTrap HP column (Cytiva) equilibrated in PBS with 0.1% LDAO.The column was washed with 20 column volumes of buffer consisting of PBS, 300 mM NaCl, 30 mM imidazole pH 7.0 and 0.45% 1-O-(n-octyl)-tetraethyleneglycol (C 8 E 4 ) to exchange the detergent.OmpW KP was eluted in buffer consisting of 250 mM imidazole and 0.45% C 8 E 4 .OmpW KP was then exchanged into 50 mM NaCl, 10 mM HEPES pH 7.0 and 0.45% C 8 E 4 using a PD-10 Desalting Column (Cytiva) and concentrated to 15 mg ml À 1 .Macromolecule-production information is summarized in Table 1.

Crystallization
Purified OmpW KP underwent preliminary screening by the sitting-drop vapour-diffusion method at 293 K using the sparse-matrix MemGold screen (Molecular Dimensions).The protein was mixed with the precipitant in a 1:1 ratio using a Mosquito LCP crystallization robot (SPT Labtech).Orthorhombic crystals appeared after 24 h in the following condition: 0.35 M lithium sulfate, 0.1 M sodium acetate pH 4.0, 11% PEG 600.Large OmpW KP crystals were obtained by the hanging-drop vapour-diffusion method.Crystals were cryoprotected in a mixture of well solution supplemented with 30% PEG 600.

Data collection and processing
Diffraction data were collected on the I03 beamline at Diamond Light Source (DLS), Didcot, United Kingdom using an EIGER2 XE 16M detector.The crystals belonged to space group C222.Diffraction frames were indexed and integrated using the DIALS pipeline as implemented at DLS (Winter et al., 2018).The data were scaled using AIMLESS in the CCP4 suite (Evans & Murshudov, 2013;Agirre et al., 2023).The data-collection parameters and merging statistics are summarized in Table 2.

Structure solution, model building and refinement
The structure of OmpW KP was solved by molecular replacement with the AlphaFold-predicted model of OmpW KP (Jumper et al., 2021) using Phenix (Liebschner et al., 2019).The calculated Matthews coefficient (V M ) was 3.84 A ˚3 Da À 1 , suggesting the presence of one molecule of OmpW KP in the asymmetric unit; this corresponds to a solvent content of 68% by volume.Manual adjustments to the model were performed in Coot (Emsley et al., 2010).Density for two sulfate ions was present and they were included in the model.Phenix was used for refinement (Afonine et al., 2018).MolProbity was used for validation (Williams et al., 2018).

Purification and crystallization of OmpW KP
OmpW KP was overexpressed in E. coli and purified in C 8 E 4 to homogeneity by immobilized metal affinity chromatography.OmpW KP displays a monodisperse peak on sizeexclusion chromatography and was >95% pure as judged by SDS-PAGE (Fig. 1a).OmpW a solution consisting of 0.35 M lithium sulfate, 0.1 M sodium acetate pH 4.0, 11%(w/v) PEG 600 (Fig. 1b).The crystals had an orthorhombic shape and were further optimized by the hanging-drop vapour-diffusion method.The optimized crystals diffracted X-rays to 3.2 A ˚resolution and belonged to space group C222.

Structure solution of OmpW KP
The structure of OmpW KP was solved by molecular replacement using the AlphaFold-predicted model.Continuous electron density could be observed for most of the structure except for Gly41-Phe52, which were omitted from model building.The OmpW KP structure consists of eight antiparallel �-strands (�1-�8) that arrange to form a hollow �-barrel in the OM and an extracellular solvent-exposed region (Fig. 2a).The extracellular region is formed from the extended �-strands of the barrel and a single �-helical turn (�1) connecting �5 and �6.A hydrophobic gate is present midway through the channel consisting of residues Leu89 and Trp188, as in OmpW EC (Hong et al., 2006), where the extracellular entrance to the channel is lined with hydrophobic residues (Fig. 2b).

Comparison of OmpW KP with OmpW EC
The closest structural homologue to OmpW KP is OmpW EC , which shares 82.7% sequence identity and 88% sequence similarity (Fig. 3a).The two structures can be superimposed with an r.m.s.d. of 0.54 A ˚over 171 C � atoms (Fig. 3b); they show high structural conservation of the �-barrel, with minor differences confined to the extracellular region, which displays some flexibility.The extracellular loop 1 that connects �1 and �2 is missing in both the OmpW KP and the OmpW EC structures, suggesting a highly flexible structure.This flexibility could be associated with substrate recruitment, as the conformation of the modelled loop 1 blocks the channel in the AlphaFold-predicted structure.In the OmpW EC structure an LDAO molecule is bound at the extracellular region but loop 1 is not fully resolved, suggesting that the inherited flexibility cannot be stabilized upon its binding (Hong et al., 2006).This highly mobile structural element on the extracellular loop is likely to shield the hydrophobic face of the extracellular region and it could transiently open to recruit hydrophobic substrates.Despite the sequence conservation of loop 1 being low between OmpW KP and OmpW EC , this suggests that it might be involved in substrate selectivity between different bacterial species.
Despite amino-acid differences in the extracellular region between OmpW KP and OmpW EC (Fig. 3c), where the tip of TraN R100-1 has been shown to bind (Low et al., 2023), binding of TraN R100-1 is not impaired between the two species.We previously reported that Ala142, which is conserved between OmpW KP and OmpW EC , acts as the minimum residue for specificity towards TraN R100-1 (Low et al., 2023); the equivalent residue in Citrobacter rodentium OmpW (OmpW CR ) is Asn142, which prevents R100-1 conjugation because of a steric clash with the tip of TraN R100-1 (Low et al., 2023).The N142A mutation in OmpW CR restored conjugation efficiency (Low et al., 2023).
In conclusion, we have resolved the crystal structure of OmpW KP ; structural comparison with OmpW EC identified the presence of a highly flexible loop, loop 1, that might be important for shielding the pore prior to hydrophobic substrate recruitment.In addition, despite sequence and structural differences in the extracellular region, both porins can mediate interactions with TraN�.

Figure 1
Figure 1 Purification and crystallization of OmpW KP .(a) SEC analysis of OmpW KP shows a monodisperse peak, with SDS-PAGE analysis of purified OmpW KP ; the purity is greater than 95%.(b) Orthorhombic OmpW KP crystals.The largest crystals had dimensions of 100 � 20 � 20 mm.

Figure 2
Figure 2 Structure of OmpW KP .(a) Cartoon representation of the OmpW KP structure (shown in green) perpendicular to the OM (depicted in grey).Sulfate ions are depicted as sticks (O atoms are shown in red and S atoms in yellow).The missing residues are marked with a green dashed line.(b) The hydrophobic residues lining the extracellular region and forming the hydrophobic gate, Leu89 and Trp188, are shown as orange sticks.

Figure 3
Figure 3Sequence alignment and superimposition of OmpW KP with OmpW EC .(a) A sequence alignment of OmpW EC (UniProt ID P0A915) and OmpW KP (UniProt ID W9B759) is shown; conserved and similar residues are shown in red and blue boxes, respectively.Residue numbers are indicated above the protein sequences.An asterisk indicates the mature protein after cleavage of the signal peptide.The alignment was prepared using ESPript(Robert & Gouet, 2014).(b) OmpW KP (green) superimposed with OmpW EC (grey; PDB entry 2f1v;Hong et al., 2006) shows high structural conservation.The LDAO molecule bound to OmpW EC is shown as sticks.(c) Close-up view of the extracellular regions of OmpW KP (green) and OmpW EC (grey), with the side chains of amino-acid differences shown as stick models.The conserved Ala142 is shown in magenta.

Table 1
OmpW KP construct design.The NcoI restriction site is underlined.‡ The XhoI restriction site is underlined.x The pTAMA signal sequence that is not present after cleavage is underlined.

Table 3
Structure solution and refinement.Values in parentheses are for the outer shell.

Table 2
Data collection and processing.