Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor

MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton flow with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotB(C)). MotB(C) significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotB(C)(Delta197-210), showed a significantly reduced inhibitory effect. Wild-type MotB(C) and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotB(C)-His(6) by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB(C) heterodimer formation in the periplasm. However, the monomeric mutant variant MotB(C)(Delta197-210) did not bind to MotB, suggesting that MotB(C) is directly involved in stator assembly. We propose that the MotB(C) dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.     

Characterization of the periplasmic region of PomB, a Na+-driven flagellar stator protein in Vibrio alginolyticus

The stator proteins PomA and PomB form a complex that couples Na⁺ influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB_C). To investigate the function of PomB_C, a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB_C fragment consisting of residues 135 to 315 (PomB_C5) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB_ΔL) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na⁺ influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB_ΔL stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal α-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a “plug” segment is important for the control of Na⁺ influx through the stator complex as well as for stator assembly.     

Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor

MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFP-FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotA(C) ), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotA(C) with FliG is required for the efficient assembly of the stators around the rotor.     

Ion-coupling determinants of Na+-driven and H+-driven flagellar motors

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7(E)) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7(E)) supported sodium-driven motility in motA or motB E.coli cells, and the swimming speed was even higher than with the original stator of E.coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7(E)) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor.     

Load‐sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor

The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild‐type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild‐type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their ‘unplugged’ forms were only half of the conductivity of the wild‐type channel. These results suggest that the D33E mutation induces a load‐dependent inactivation of the MotA/B complex. We propose that the stator element is a load‐sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co‐ordinated proton translocation.     

Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation

The bacterial flagellar motor is driven by an ion flux through a channel called MotAB in Escherichia coli or Salmonella and PomAB in Vibrio alginolyticus. PomAB is composed of two transmembrane (TM) components, PomA and PomB, and converts a sodium ion flux to rotation of the flagellum. Its homolog, MotAB, utilizes protons instead of sodium ions. PomB/MotB has a peptidoglycan (PG)-binding motif in the periplasmic domain, allowing it to function as the stator by being anchored to the PG layer. To generate torque, PomAB/MotAB is thought to undergo a conformational change triggered by the ion flux and to interact directly with FliG, a component of the rotor. Here, we present the first three-dimensional structure of this torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the TM and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a "plugged" conformation, which would prevent ions leaking through the channel. We propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators.     

The Escherichia coli MotAB proton channel unplugged

The MotA and MotB proteins of Escherichia coli serve two functions. The MotA4MotB2 complex attaches to the cell wall via MotB to form the stator of the flagellar motor. The complex also couples the flow of hydrogen ions across the cell membrane to movement of the rotor. The TM3 and TM4 transmembrane helices of MotA and the single TM of MotB comprise the proton channel, which is inactive until the complex assembles into a motor. Here, we identify a segment of the MotB protein that acts as a plug to prevent premature proton flow. The plug is in the periplasm just C-terminal to the MotB TM. It consists of an amphipathic alpha helix flanked by Pro52 and Pro65. When MotA is over-expressed with MotB deleted for residues 51-70, a massive influx of protons acidifies the cytoplasm without significantly depleting the proton motive force. Either that acidification or some sequela thereof, such as potassium or water efflux from the cells, inhibits growth. The Pro residues and Ile58, Tyr61, and Phe62 are essential for plug function. Cys-substituted MotB proteins form a disulfide bond between the two plugs that hold the channels open, and the plugs function intrans within the MotA4MotB2 complex. We present a model in which the MotA4MotB2 complex forms in the bulk membrane. Before association with a motor, we propose the plugs insert into the cell membrane parallel with its periplasmic face and interfere with channel formation. When a complex incorporates into a motor, the plugs leave the membrane and associate with each other via their hydrophobic faces to hold the proton channel open.     

Suppressor analysis of the MotB(D33E) mutation to probe bacterial flagellar motor dynamics coupled with proton translocation

MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.     

Structural and functional characterization of SiiA,an auxiliary protein from the SPI4‐encoded type 1 secretion system from Salmonella enterica

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)‐encoded type one secretion system (T1SS) and the SPI1‐encoded type three secretion system (T3SS‐1). The SPI4‐encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure–function relationships in SiiA, a non‐canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA‐PD). The crystal structure of SiiA‐PD displays homology to that of MotB and other peptidoglycan (PG)‐binding domains. SiiA‐PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA‐MotB homology is paralleled by a predicted SiiB‐MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.     

Structure and Function of the Escherichia coli Tol-Pal Stator Protein TolR

TolR is a 15-kDa inner membrane protein subunit of the Tol-Pal complex in Gram-negative bacteria, and its function is poorly understood. Tol-Pal is recruited to cell division sites where it is involved in maintaining the integrity of the outer membrane. TolR is related to MotB, the peptidoglycan (PG)-binding stator protein from the flagellum, suggesting it might serve a similar role in Tol-Pal. The only structure thus far reported for TolR is of the periplasmic domain from Haemophilus influenzae in which N- and C-terminal residues had been deleted (TolR(62–133), Escherichia coli numbering). H. influenzae TolR(62–133) is a symmetrical dimer with a large deep cleft at the dimer interface. Here, we present the 1.7-Å crystal structure of the intact periplasmic domain of E. coli TolR (TolR(36–142)). E. coli TolR(36–142) is also dimeric, but the architecture of the dimer is radically different from that of TolR(62–133) due to the intertwining of its N and C termini. TolR monomers are rotated ∼180° relative to each other as a result of this strand swapping, obliterating the putative PG-binding groove seen in TolR(62–133). We found that removal of the strand-swapped regions (TolR(60–133)) exposes cryptic PG binding activity that is absent in the full-length domain. We conclude that to function as a stator in the Tol-Pal complex dimeric TolR must undergo large scale structural remodeling reminiscent of that proposed for MotB, where the N- and C-terminal sequences unfold in order for the protein to both reach and bind the PG layer ∼90 Å away from the inner membrane.     

Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes

A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.     

The role of a cytoplasmic loop of MotA in load‐dependent assembly and disassembly dynamics of the MotA/B stator complex in the bacterial flagellar motor

The proton‐driven flagellar motor of Salmonella enterica can accommodate a dozen MotA/B stators in a load‐dependent manner. The C‐terminal periplasmic domain of MotB acts as a structural switch to regulate the number of active stators in the motor in response to load change. The cytoplasmic loop termed MotA_C is responsible for the interaction with a rotor protein, FliG. Here, to test if MotA_C is responsible for stator assembly around the rotor in a load‐dependent manner, we analyzed the effect of MotA_C mutations, M76V, L78W, Y83C, Y83H, I126F, R131L, A145E and E155K, on motor performance over a wide range of external load. All these MotA_C mutations reduced the maximum speed of the motor near zero load, suggesting that they reduce the rate of conformational dynamics of MotA_C coupled with proton translocation through the MotA/B proton channel. Dissociation of the stators from the rotor by decrease in the load was facilitated by the M76V, Y83H and A145E mutations compared to the wild‐type motor. The E155K mutation reduced the number of active stators in the motor from 10 to 6 under extremely high load. We propose that MotA_C is responsible for load‐dependent assembly and disassembly dynamics of the MotA/B stator units.     

Crystallographic and molecular dynamics analysis of loop motions unmasking the peptidoglycan-binding site in stator protein MotB of flagellar motor

Background

The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues.

Methodology/Principal Findings

We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan.

Conclusion/Significance

Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall.     

Solubilization and purification of the MotA/MotB complex of Escherichia coli

Bacterial flagella are driven at their base by a rotary motor fueled by the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which function together to conduct ions across the membrane and couple ion flow to rotation. An invariant aspartate residue in MotB (Asp32 in the protein of E. coli) is essential for rotation and appears to have a direct role in proton conduction. A recent study showed that changes at Asp32 in MotB cause a conformational change in the complex, as evidenced by altered patterns of protease susceptibility of MotA [Kojima, S., and Blair, D. F. (2001) Biochemistry 40 (43), 13041-13050]. It was proposed that protonation/deprotonation of Asp32 might regulate a conformational change in the stator that acts as the powerstroke to drive rotation of the rotor. Biochemical studies of the MotA/MotB complex have been hampered by the absence of a suitable assay for its integrity in detergent solution. Here, we have studied the behavior of the MotA/MotB complex in a variety of detergents, making use of the protease-susceptibility assay to monitor its integrity. Among about 25 detergents tested, a few were found to solubilize the proteins effectively while preserving certain conformational properties characteristic of an intact complex. The detergent dodecylphosphocholine, or DPC, proved especially effective. MotA/MotB complexes purified in DPC migrate with an apparent size of approximately 300 kDa in gel-filtration columns, and retain the Asp32-modulated conformational differences seen in membranes. (35)S-radiolabeling showed that MotA and MotB are present in a 2:1 ratio in the complex. Purified MotA/MotB complexes should enable in vitro study of the proton-induced conformational change and other aspects of stator function.     

Rusty, jammed, and well-oiled hinges: Mutations affecting the interdomain region of FliG, a rotor element of the Escherichia coli flagellar motor

The FliG protein is a central component of the bacterial flagellar motor. It is one of the first proteins added during assembly of the flagellar basal body, and there are 26 copies per motor. FliG interacts directly with the Mot protein complex of the stator to generate torque, and it is a crucial player in switching the direction of flagellar rotation from clockwise (CW) to counterclockwise and vice versa. A primarily helical linker joins the N-terminal assembly domain of FliG, which is firmly attached to the FliF protein of the MS ring of the basal body, to the motility domain that interacts with MotA/MotB. We report here the results of a mutagenic analysis focused on what has been called the hinge region of the linker. Residue substitutions in this region generate a diversity of phenotypes, including motors that are strongly CW biased, infrequent switchers, rapid switchers, and transiently or permanently paused. Isolation of these mutants was facilitated by a "sensitizing" mutation (E232G) outside of the hinge region that was accidentally introduced during cloning of the chromosomal fliG gene into our vector plasmid. This mutation partially interferes with flagellar assembly and accentuates the defects associated with mutations that by themselves have little phenotypic consequence. The effects of these mutations are analyzed in the context of a conformational-coupling model for motor switching and with respect to the structure of the C-terminal 70% of FliG from Thermotoga maritima.     

Conformational change in the stator of the bacterial flagellar motor.

MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.     

Membrane segment organization in the stator complex of the flagellar motor: implications for proton flow and proton-induced conformational change

MotA and MotB are membrane proteins that form the stator of the bacterial flagellar motor. Each motor contains several MotA 4MotB 2 complexes, which function independently to conduct protons across the membrane and couple proton flow to rotation. The mechanism of rotation is not understood in detail but is thought to involve conformational changes in the stator complexes driven by proton association/dissociation at a critical Asp residue of MotB (Asp 32 in the protein of Escherichia coli). MotA has four membrane segments and MotB has one. Previous studies using targeted disulfide cross-linking showed that the membrane segments of the two MotB subunits are together at the center of the complex, surrounded by the TM3 and TM4 segments of the four MotA subunits. Here, the cross-linking studies are extended to TM1 and TM2 of MotA, using Cys residues introduced in several positions in the segments. The observed patterns of disulfide cross-linking indicate that the TM2 segment is positioned between segments TM3 and TM4 of the same subunit, where it could contribute to the proton-channel-forming part of the structure. TM1 is at the interface between TM4 of its own subunit and the TM3 segment of another subunit, where it could stabilize the complex. A structural model based on the cross-linking results shows unobstructed pathways reaching from the periplasm to the Asp 32 residues near the inner ends of the MotB segments. The model indicates a close proximity for certain conserved, functionally important residues. The results are used to develop an explicit model for the proton-induced conformational change in the stator.     

Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors

The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H⁺ or Na⁺. The motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na⁺ motor of Vibrio alginolyticus , is thought to be a torque generator converting the energy of ion flux into mechanical power. We found that specific mutations in PomB, including D24N, F33C and S248F, which caused motility defects, affected the assembly of stator complexes into the polar flagellar motor using green fluorescent protein-fused stator proteins. D24 of PomB is the predicted Na⁺-binding site. Furthermore, we demonstrated that the coupling ion, Na⁺, is required for stator assembly and that phenamil (an inhibitor of the Na⁺-driven motor) inhibited the assembly. Carbonyl cyanide m -chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in this organism at neutral pH, also inhibited the assembly. Thus we conclude that the process of Na⁺ influx through the channel, including Na⁺ binding, is essential for the assembly of the stator complex to the flagellar motor as well as for torque generation.     

Peptidoglycan recognition by Pal, an outer membrane lipoprotein

Peptidoglycan-associated lipoprotein (Pal) is a potential vaccine candidate from Haemophilus influenzae that is highly conserved in Gram-negative bacteria and anchored to the outer membrane through an N-terminal lipid attachment. Pal stabilizes the outer membrane by providing a noncovalent link to the peptidoglycan (PG) layer through a periplasmic domain. Using NMR spectroscopy, we determined the three-dimensional structure of a complex between the periplasmic domain of Pal and a biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-alpha-d-Glu-m-Dap-D-Ala-d-Ala (m-Dap is meso-diaminopimelate). Pal has a binding pocket lined with conserved surface residues that interacts exclusively with the peptide portion of the ligand. The m-Dap residue, which is mainly found in the cell walls of Gram-negative bacteria, is sequestered in this pocket and plays an important role by forming hydrogen bond and hydrophobic contacts to Pal. The structure provides insight into the mode of cell wall recognition for a broad class of Gram-negative membrane proteins, including OmpA and MotB, which have peptidoglycan-binding domains homologous to that of Pal.