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• W2951966558 abstract "•TolR binds peptidoglycan cell wall via electrostatic interactions•Binding of TolR and OmpA keeps the cell wall flat•Closed-state TolR does not interact with the cell wall•Cell-wall-binding residues of TolR conserved across species We present a molecular modeling and simulation study of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane, in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed binds peptidoglycan largely through electrostatic interactions. Non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides, respectively, maintain the position of the cell wall even in the absence of Braun's lipoprotein. The charged residues that mediate the cell-wall interactions of TolR in our simulations are conserved across a number of species of gram-negative bacteria. We present a molecular modeling and simulation study of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane, in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed binds peptidoglycan largely through electrostatic interactions. Non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides, respectively, maintain the position of the cell wall even in the absence of Braun's lipoprotein. The charged residues that mediate the cell-wall interactions of TolR in our simulations are conserved across a number of species of gram-negative bacteria. Gram-negative bacteria such as E. coli have a complex cell envelope, which protects the cell and controls influx/efflux of molecular species to ensure the normal functioning of the cell (Nikaido, 2003Nikaido H. Molecular basis of bacterial outer membrane permeability revisited.Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2850) Google Scholar). The cell envelope contains an aqueous region known as the periplasm, which is sandwiched between an asymmetrical outer membrane and a symmetrical inner membrane. Contained within the periplasm is the cell wall, which is composed of a sugar-peptide polymer known as peptidoglycan (PGN) (Vollmer and Bertsche, 2008Vollmer W. Bertsche U. Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli.Biochim. Biophys. Acta Biomembr. 2008; 1778: 1714-1734Crossref PubMed Scopus (303) Google Scholar). The periplasm is host to many different proteins that are essential for the healthy growth and proliferation of gram-negative bacteria. These proteins are known to be freely moving, associated with the inner or outer membrane, or bound to the cell wall. The interactions of these proteins with one another both (1) laterally, in other words within one membrane or the periplasm, and (2) across regions, e.g., extending from one membrane to the periplasm, are important in maintaining the structural integrity and correct functioning of the cell envelope. A number of different proteins have been shown to play a role in cross-region interactions, and others have been hypothesized to do so. Braun's lipoprotein (BLP; also known as “Lpp” and “murein lipoprotein”) is an abundant protein that is lipidated at its N-terminal domain, which anchors it to the outer membrane (Braun, 1975Braun V. Covalent lipoprotein from outer membrane of Escherichia-coli.Biochim. Biophys. Acta. 1975; 415: 335-377Crossref PubMed Scopus (362) Google Scholar). It is the only known protein in E. coli to be covalently attached to the PGN of the cell wall. It exists in two states: ∼33% of the lipoprotein is covalently bound to the cell wall via a peptide bond, and ∼66% is free in the periplasm. BLP is proposed to have a primarily structural function, essentially acting as a staple between the outer membrane and the PGN, which serves to maintain the required distance between the cell wall and the outer membrane (Miller and Salama, 2018Miller S.I. Salama N.R. The gram-negative bacterial periplasm: size matters.PLoS Biol. 2018; 16: e2004935Crossref PubMed Scopus (71) Google Scholar). Cells that lack BLP or that have reduced amounts of BLP are viable, but they have been shown to release outer-membrane vesicles at a higher rate than normal and also suffer from cellular leakage (Schwechheimer et al., 2014Schwechheimer C. Kulp A. Kuehn M.J. Modulation of bacterial outer membrane vesicle production by envelope structure and content.BMC Microbiol. 2014; 14: 324Crossref PubMed Scopus (100) Google Scholar, Asmar and Collet, 2018Asmar A.T. Collet J.F. Lpp, the Braun lipoprotein, turns 50—major achievements and remaining issues.FEMS Microbiol. Lett. 2018; 365https://doi.org/10.1093/femsle/fny199Crossref PubMed Scopus (51) Google Scholar). Non-covalent interactions between the cell wall and the outer membrane are mediated through proteins such as PAL and OmpA (Parsons et al., 2006Parsons L.M. Lin F. Orban J. Peptidoglycan recognition by Pal, an outer membrane lipoprotein.Biochemistry. 2006; 45: 2122-2128Crossref PubMed Scopus (134) Google Scholar, Park et al., 2012Park J.S. Lee W.C. Yeo K.J. Ryu K.S. Kumarasiri M. Hesek D. Lee M. Mobashery S. Song J.H. Il Kim S. et al.Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane.FASEB J. 2012; 26: 219-228Crossref PubMed Scopus (121) Google Scholar). The latter is composed of two domains, the N-terminal domain, which is an eight-stranded β barrel that is connected via a flexible linker to the soluble C-terminal domain, which contains the PGN-binding region (Carpenter et al., 2007Carpenter T. Khalid S. Sansom M.S. A multidomain outer membrane protein from Pasteurella multocida: modelling and simulation studies of PmOmpA.Biochim. Biophys. Acta. 2007; 1768: 2831-2840Crossref PubMed Scopus (15) Google Scholar, Marcoux et al., 2014Marcoux J. Politis A. Rinehart D. Marshall D.P. Wallace M.I. Tamm L.K. Robinson C.V. Mass spectrometry defines the C-terminal dimerization domain and enables modeling of the structure of full-length OmpA.Structure. 2014; 22: 781-790Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). We have previously shown that OmpA in its dimeric form can extend its linker region such that the C-terminal domain is able to form long-lasting interactions with PGN even in the absence of BLP, while BLP facilitates PGN binding of the monomer (Samsudin et al., 2017Samsudin F. Boags A. Piggot T.J. Khalid S. Braun's lipoprotein facilitates OmpA interaction with the Escherichia coli cell wall.Biophys. J. 2017; 113: 1496-1504Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). We showed that BLP can tilt within the periplasm to provide some variation in the PGN-outer membrane distance. Interactions of inner membrane proteins with the cell wall are less well understood at the molecular level than their outer-membrane counterparts. Three proteins from the Tol family, TolQ, TolR, and TolA, interact with one another via their transmembrane domains within the inner membrane (Gerding et al., 2007Gerding M.A. Ogata Y. Pecora N.D. Niki H. De Boer P.A.J. The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli.Mol. Microbiol. 2007; 63: 1008-1025Crossref PubMed Scopus (267) Google Scholar). TolR is proposed to interact with the cell wall in a manner similar to that of OmpA. While binding of TolR to PGN has been demonstrated, the X-ray structure of TolR from E. coli is of the protein in its compact form, in which the putative PGN-binding domain is not surface exposed (Wojdyla et al., 2015Wojdyla J.A. Cutts E. Kaminska R. Papadakos G. Hopper J.T. Stansfeld P.J. Staunton D. Robinson C.V. Kleanthous C. Structure and function of the Escherichia coli Tol-Pal stator protein TolR.J. Biol. Chem. 2015; 290: 26675-26687Crossref PubMed Scopus (18) Google Scholar). Based on the X-ray structure of the closed state and biophysical and computational studies of TolR, Kleanthous and co-workers proposed a large-scale proton-motive force (PMF)-dependent conformational rearrangement in which extension of the TolR linker enables the protein to contact the cell wall and exposure of the PGN-binding domain enables it to bind PGN in a manner similar to the structural alterations proposed for the bacterial flagellar protein MotB (Wojdyla et al., 2015Wojdyla J.A. Cutts E. Kaminska R. Papadakos G. Hopper J.T. Stansfeld P.J. Staunton D. Robinson C.V. Kleanthous C. Structure and function of the Escherichia coli Tol-Pal stator protein TolR.J. Biol. Chem. 2015; 290: 26675-26687Crossref PubMed Scopus (18) Google Scholar). The model of the protein in this conformation was termed the “open state.” The hypothesis of large-scale rearrangement is difficult to test experimentally in the absence of structures of the different states of the proteins. However, simulations offer a route to predict the behavior of the model under different scenarios. In the last decade or so, molecular dynamics studies of the cell envelopes of gram-negative bacteria have moved beyond simple phospholipid representations of both envelopes, to incorporate the natural biochemical diversity of the lipidic components of these membranes, at both atomistic (Kirschner et al., 2012Kirschner K.N. Lins R.D. Maass A. Soares T.A. A glycam-based force field for simulations of lipopolysaccharide membranes: Parametrization and validation.J. Chem. Theory Comput. 2012; 8: 4719-4731Crossref PubMed Scopus (83) Google Scholar, Piggot et al., 2011Piggot T.J. Holdbrook D.A. Khalid S. Electroporation of the E. coli and S. Aureus membranes: molecular dynamics simulations of complex bacterial membranes-suppinfo.J. Phys. Chem. 2011; Crossref Scopus (174) Google Scholar, Wu et al., 2013Wu E.L. Engström O. Jo S. Stuhlsatz D. Yeom M.S. Klauda J.B. Widmalm G. Im W. Molecular dynamics and NMR spectroscopy studies of E. coli lipopolysaccharide structure and dynamics.Biochem. J. 2013; 105: 1444-1455Scopus (139) Google Scholar) and coarse-grain resolution (Hsu et al., 2016Hsu P.-C. Jefferies D. Khalid S. Molecular Dynamics Simulations Predict the Pathways via Which Pristine Fullerenes Penetrate Bacterial Membranes.J. Phys. Chem. B. 2016; 120: 11170-11179Crossref PubMed Scopus (46) Google Scholar, Ma et al., 2015Ma H. Irudayanathan F.J. Jiang W. Nangia S. Simulating gram-negative bacterial outer membrane: a coarse grain model.J. Phys. Chem. B. 2015; 119: 14668-14682Crossref PubMed Scopus (63) Google Scholar). Much of the setup of such systems is facilitated by tools such MARTINI-MAKER (Hsu et al., 2017Hsu P.C. Bruininks B.M.H. Jefferies D. Cesar Telles de Souza P. Lee J. Patel D.S. Marrink S.J. Qi Y. Khalid S. Im W. CHARMM-GUI Martini Maker for modeling and simulation of complex bacterial membranes with lipopolysaccharides.J. Comp. Chem. 2017; 38: 2354-2363Crossref PubMed Scopus (93) Google Scholar). Furthermore, detailed atomistic models of the cell wall have recently emerged, too, enabling study of the biophysical properties of PGN (Gumbart et al., 2014Gumbart J.C. Beeby M. Jensen G.J. Roux B. Escherichia coli Peptidoglycan Structure and Mechanics as Predicted by Atomic-Scale Simulations.PLoS Comput. Biol. 2014; 10Crossref PubMed Scopus (70) Google Scholar, Hwang et al., 2018Hwang H. Paracini N. Parks J.M. Lakey J.H. Gumbart J.C. Distribution of mechanical stress in the Escherichia coli cell envelope.Biochim. Biophys. Acta. 2018; 1860: 2566-2575Crossref Scopus (49) Google Scholar) and interactions with proteins (Samsudin et al., 2016Samsudin F. Ortiz-Suarez M.L. Piggot T.J. Bond P.J. Khalid S. OmpA: a flexible clamp for bacterial cell wall attachment.Structure. 2016; 24: 2227-2235Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To test the model of the open state of TolR and compare the PGN-binding mode with that of OmpA, in the following we present an atomistic molecular dynamics and modeling study of TolR, OmpA, and BLP in a model of the cell envelope that includes both membranes and the cell wall. We note here that the structures of TolQ and TolA are not known, neither are the precise details of the way they are arranged with respect to each other and TolR; thus TolA and TolQ are omitted from the present studies. We show that the model of the open state of TolR binds PGN primarily through electrostatic interactions, whereas the closed state does not bind PGN. In the presence of full-length OmpA dimers in the outer membrane and open-state TolR in the inner membrane, the location of the cell wall is maintained between these proteins. The binding of both proteins to the cell wall also alleviates local surface distortions that are observed when only one of OmpA or TolR is bound. In contrast, if OmpA is truncated to its N-terminal domain and BLP is added to the system, then the TolR linker is able to contract and, in doing so, “pulls” the cell wall down toward the inner membrane until BLP is fully stretched and further movement is not possible. For ease of interpretation of the results the simulations described below are summarized in Table 1. The simulations of TolR and OmpA were performed with a monolayered cell wall. The reason for this is that, from test simulations of one to three layers of cell wall, we observed the thickness of three layers to be 90–100 Å, two layers to be 60–70 Å, and a single layer to be ∼30 Å (Figure S1), and given that the proposed thickness of PGN in E. coli is 20–70 Å (Matias et al., 2003Matias V.R.F. Al-amoudi A. Dubochet J. Beveridge T.J. Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa.J. Bacteriol. 2003; 185: 6112-6118Crossref PubMed Scopus (271) Google Scholar, Turner et al., 2013Turner R.D. Hurd A.F. Cadby A. Hobbs J.K. Foster S.J. Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture.Nat. Commun. 2013; 4: 1496-1498Crossref PubMed Scopus (96) Google Scholar), a single layer was chosen.Table 1Summary of SimulationsOmpA StructureTolR StructureBLPMembraneNumber of AtomsBinds to PGNSimulation Length (ns)Noneopennoneinner only135,718yes3 × 200Wild-typeopennoneinner and outer207,787yes3 × 200Wild-typeopenyesinner and outer275,948yes3 × 2001-Truncatedopenyesinner and outer238,293yes3 × 200Truncatedopenyesinner and outer236,933yes4 × 200Truncatedclosedyesinner and outer236,789no3 × 200 Open table in a new tab In simulations of TolR and full-length OmpA, both proteins were initially positioned either directly in contact with or close to the cell wall. Specifically, the OmpA-PGN complex was taken from our previous work and the TolR was positioned with the transmembrane helices embedded in the inner membrane, and the periplasmic domain was not in contact with the cell wall (Samsudin et al., 2017Samsudin F. Boags A. Piggot T.J. Khalid S. Braun's lipoprotein facilitates OmpA interaction with the Escherichia coli cell wall.Biophys. J. 2017; 113: 1496-1504Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The shortest distance between the TolR periplasmic domain and the cell wall was around 5 Å at the start of the simulation. This system configuration gives a periplasmic space width of around 170 Å (experimental estimates of the width vary between 100 and 250 Å; Graham et al., 1991Graham L.L. Beveridge T.J. Nanninga N. Periplasmic space and the concept of the periplasm.Trends Biochem. Sci. 1991; 16: 328-329Abstract Full Text PDF PubMed Scopus (81) Google Scholar, Vollmer and Seligman, 2010Vollmer W. Seligman S.J. Architecture of peptidoglycan: more data and more models.Trends in Microbiol. 2010; 18: 59-66Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The periplasmic domain of TolR in the open conformation was structurally stable in all simulations and showed similar root-mean-square deviation (RMSD) progressions compared with the C-terminal domain of OmpA (Figure S2). The secondary structure of this domain was also largely preserved during the simulations. In this state TolR has a long unstructured loop in the C terminus, which remained mobile throughout the simulations. In all simulations of the wild-type TolR in the open state, binding to PGN was observed. The mechanism of cell-wall binding proceeded as follows: the proline (Pro141) residues at the C terminus of TolR consistently formed the first contact with PGN via its carboxyl group that interacted with either the positively charged amine group of diaminopimelate (mDAP) or the polar amide and hydroxyl moieties in adjacent sugars (Figures 1 and S3). This was immediately followed by interactions with downstream polar residues (Thr139 and Gln140). The greater flexibility of the unstructured C-terminal loop enabled this initial binding process as these residues were able to “snorkel” toward the PGN. Glu89 and Lys122, found in the more rigid globular domain of TolR, strengthened this binding; the former interacted with hydroxyl groups in N-acetylmuramic acid, while the latter formed a salt bridge with the C terminus of mDAP. Within about 10 ns, in each simulation, the TolR linker was extended such that the periplasmic domain was in contact with PGN; in other words, PGN binding had occurred. Across all independent repeat simulations, after 200 ns, the cell wall was located about 20 Å (along the z direction, perpendicular to the plane of the membrane) from each protein, reaching a stable position after ∼100 ns of simulation (Figures 2A and 2B ). We extended two of these simulations to 500 ns; the binding of OmpA and TolR to the cell wall was maintained (Figure S4). The linker regions of both proteins were only partially extended to enable the cell wall to be maintained at this position. In other words, the proteins had the potential to adopt other arrangements in terms of their location with respect to the cell wall but maintained a position in which the cell was sandwiched equidistant between the two proteins for the duration of these simulations. Given we have previously reported details of the interactions between OmpA and PGN (Samsudin et al., 2016Samsudin F. Ortiz-Suarez M.L. Piggot T.J. Bond P.J. Khalid S. OmpA: a flexible clamp for bacterial cell wall attachment.Structure. 2016; 24: 2227-2235Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, Samsudin et al., 2017Samsudin F. Boags A. Piggot T.J. Khalid S. Braun's lipoprotein facilitates OmpA interaction with the Escherichia coli cell wall.Biophys. J. 2017; 113: 1496-1504Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), here we focus on the details of the TolR-PGN interactions. Analysis of the TolR residues in contact with PGN (where contact is defined as an interatomic distance of ≤4 Å) reveals the five residues that made frequent contacts. These are Glu89, Lys122, Thr139, Gln140, and Pro141 (Figure 2C), and they are the same residues identified above as being key to the initial binding and stabilization process. More specifically, the arrangement of PGN in the cell wall of E. coli is such that numerous hydroxyl and amide groups on the sugar backbone and the peptide chains are available for hydrogen bonds (Figure 3A). In addition, there are three negatively charged carboxyl groups and one positively charged amine group on the non-cross-linked peptide chains (on residue D-glutamate, residue D-alanine, and mDAP) that can form salt bridges with TolR periplasmic domain. Examples of these interactions involving the key residues identified in Figure 2C are shown in Figure 3B. We found 13 TolR residues that formed hydrogen bonds with the different parts of the cell wall (Figure 3C). These hydrogen bonds were short-lived, with each lasting no longer than 30% of the simulation timescale. Fewer salt bridge interactions were found, as there are only five charged residues that are accessible to the cell wall. These salt bridges, however, were longer lasting, with lifetimes up to 80% of the simulation timescale (Figure 3D). Decomposition of binding free energy shows a larger coulombic contribution compared with that of van der Waals interactions, which is concordant with the numerous hydrogen bonds and salt bridges (Figure 3E). Interestingly, the coulombic contribution of the free energy is correlated to the number of hydrogen bonds formed between TolR and the cell wall, suggesting that hydrogen bond formation is key for TolR binding (Figure S5). Mapping the electrostatic surface of TolR revealed a predominantly negatively charged surface facing the cell wall contributed by the carboxyl groups on the C terminus and downstream polar residues (Figure S6). Smaller positively charged patches are found interspersed around this negatively charged surface due to basic residues like Lys122. It makes sense, therefore, that the negatively charged C-terminal region of TolR formed initial binding to the cell wall via electrostatic interactions with positive moieties of the PGN. To quantify the forces required to maintain TolR-cell wall binding, we performed steered molecular dynamics simulations whereby the periplasmic domain of TolR was pulled away from the cell wall. An average force of around 400 kJ mol−1 nm−1 was required to detach the protein from the cell wall (Figure S7A). In comparison, a similar set of simulations performed on the OmpA periplasmic domain revealed a much higher force (∼1,500 kJ mol−1 nm−1) (Figure S7B), potentially due to the larger size of the OmpA periplasmic domain compared with that of TolR (2,490 and 1,512 atoms, respectively). OmpA therefore has a bigger surface area for interaction with the cell wall resulting in a stronger binding. To determine whether the aforementioned location of the cell wall, approximately equidistant from TolR in the inner membrane and OmpA in the outer membrane, is a consequence of the PGN binding strength of the protein domains being approximately equal, or simply a function of the starting position of the simulations, the C-terminal domain and the linker region of OmpA were truncated, leaving the N-terminal β barrel (residues 1–172). One BLP trimer was incorporated into these simulation systems to provide an anchor between the cell wall and the outer membrane. BLP is covalently attached to the cell wall via a peptide bond between the C-terminal lysine residue on one of its protomers and the mDAP moiety of the PGN. The remaining two BLP protomers are not covalently linked to the cell wall and their C-terminal lysine residues are able to form salt bridge interactions with negatively charged moieties of the PGN, hence providing additional support to the cell wall. In the full-length OmpA simulations and when only one of the OmpA protomers was truncated, the cell wall remained bound to both OmpA and TolR in a fashion similar to the wild-type simulations, while BLP tilted to around 60° with respect to the plane of the outer membrane (Figures 4 and S8). When both protomers were truncated, however, two distinct behaviors were observed. In three of the simulations TolR remained extended and bound to the cell wall, with the BLP trimer tilted at 60° to enable the location of the cell wall to remain approximately equidistant between the two membranes (Figure 5A). In contrast, in one of the four simulations, the linker of TolR contracted such that the bulk of the protein moved to rest on the inner membrane, with BLP almost at right angles to the plane of the outer membrane (Figure 5B). Interestingly, TolR remained bound to PGN throughout this process. This provides compelling evidence that the PGN-binding domain of TolR proposed by Kleanthous and co-workers does indeed stably bind PGN, and that this binding can withstand contraction of the TolR linker (Wojdyla et al., 2015Wojdyla J.A. Cutts E. Kaminska R. Papadakos G. Hopper J.T. Stansfeld P.J. Staunton D. Robinson C.V. Kleanthous C. Structure and function of the Escherichia coli Tol-Pal stator protein TolR.J. Biol. Chem. 2015; 290: 26675-26687Crossref PubMed Scopus (18) Google Scholar). Furthermore, these observations show that the balance of non-covalent interactions between proteins in both membranes and the cell wall acts like a clamp from both sides in maintaining the position of the cell wall. BLP by itself on the outer membrane side is not sufficient, given that its ability to bend and tilt enables significant deviation in the cell-wall position. This agrees with experimental studies that showed that mutations in either the tolR or the ompA gene destabilized the cell envelope, resulting in the formation of outer membrane vesicles in E. coli (Deatherage et al., 2009Deatherage B.L. Lara J.C. Bergsbaken T. Barrett S.L.R. Lara S. Cookson B.T. Biogenesis of bacterial membrane vesicles.Mol. Microbiol. 2009; 72: 1395-1407Crossref PubMed Scopus (190) Google Scholar, Perez-Cruz et al., 2016Perez-Cruz C. Canas M.A. Gimenez R. Badia J. Mercade E. Baldoma L. Aguilera L. Membrane vesicles released by a hypervesiculating Escherichia coli Nissle 1917 tolR mutant are highly heterogeneous and show reduced capacity for epithelial cell interaction and entry.PLoS One. 2016; 11: e0169186Crossref PubMed Scopus (24) Google Scholar). Our previous study showed that binding of OmpA to the cell wall caused a local buckling effect on the latter, whereby the surface of the cell wall noticeably curved toward the outer membrane at the point of contact (Samsudin et al., 2017Samsudin F. Boags A. Piggot T.J. Khalid S. Braun's lipoprotein facilitates OmpA interaction with the Escherichia coli cell wall.Biophys. J. 2017; 113: 1496-1504Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Similarly here, when only TolR and the cell wall (no OmpA or BLP) were included in the simulation system, the contraction of the linker pulled the cell wall toward the inner membrane, resulting in local curvature of the cell wall. In contrast, when both OmpA and TolR were bound to the cell wall, the degree of undulation observed during the simulations was significantly reduced (Figure 6). Interestingly the distortions were also significantly reduced when TolR was bound to the cell wall in the presence of BLP (but without OmpA in the outer membrane). This is in agreement with our previous studies of the outer membrane and the cell wall in which BLP was able to prevent undulations that were otherwise present when OmpA alone was bound to the cell wall. These observations suggest that TolR and OmpA binding to the cell wall from either side of the cell envelope prevents any local distortions caused by either one of them binding alone. From the outer membrane side, BLP also plays a role to this effect; presumably, the greater the number of membrane protein interactions with the cell wall, the less distorted the cell wall is, but this hypothesis should be tested with a wider range of PGN-binding proteins. As a further test of whether the PGN binding of TolR is specific to the identified binding domain, or whether other regions of the protein can bind PGN too, TolR-PGN interactions when TolR is in its closed conformation were also explored. The coordinates for the protein were taken from the X-ray structure (PDB: 5BY4), with the transmembrane helices modeled in as reported by Wojdyla et al., 2015Wojdyla J.A. Cutts E. Kaminska R. Papadakos G. Hopper J.T. Stansfeld P.J. Staunton D. Robinson C.V. Kleanthous C. Structure and function of the Escherichia coli Tol-Pal stator protein TolR.J. Biol. Chem. 2015; 290: 26675-26687Crossref PubMed Scopus (18) Google Scholar. The final snapshot was extracted from our simulation with truncated OmpA dimer in which the TolR linker had contracted to enable interaction of the protein with the inner membrane while still being bound to PGN. The TolR was replaced by the X-ray structure of the closed state. Thus, at the start of the simulation, BLP was extended and essentially at right angles to the plane of the outer membrane, and the TolR in the closed state was in contact with the inner membrane and within 5 Å of the cell wall (similar to our previous setup with open state TolR). After 200 ns of simulation BLP tilted to pull the cell wall approximately 20 Å toward the outer membrane and away from TolR (Figure 7). The electrostatic surface potential of the periplasmic domain facing the cell wall in the TolR closed state is similar to that in the open state, i.e., predominantly negatively charged surface surrounding small positively charged patches (Figure S6). In the closed state, however, the mobile C-terminal domain of TolR responsible for the initial interaction with the cell wall and the flexible linker connecting the periplasmic domain and the N-terminal helices are folded together into a β sheet buried within the dimeric structure. The lack of PGN binding of the protein in this state provides further evidence that binding of TolR requires specific domains that are not accessible in the closed state of the protein. Having identified Glu89 and Lys122 as key residues in the interaction of TolR and the cell wall, it is worth investigating if this binding mechanism is universally conserved. Sequence alignment of TolR from different gram-negative bacteria, as well as its structural homolog, ExbD from the TonB system, revealed that both residues are well preserved (Figure" @default.
• W2951966558 created "2019-06-27" @default.
• W2951966558 creator A5041438560 @default.
• W2951966558 creator A5074797108 @default.
• W2951966558 creator A5079670006 @default.
• W2951966558 date "2019-04-01" @default.
• W2951966558 modified "2023-09-27" @default.
• W2951966558 title "Binding from Both Sides: TolR and Full-Length OmpA Bind and Maintain the Local Structure of the E. coli Cell Wall" @default.
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