Bitopic Membrane Protein Research Articles

Phage Shock Protein C (PspC) of Yersinia enterocolitica Is a Polytopic Membrane Protein with Implications for Regulation of the Psp Stress Response

Phage shock proteins B (PspB) and C (PspC) are integral cytoplasmic membrane proteins involved in inducing the Yersinia enterocolitica Psp stress response. A fundamental aspect of these proteins that has not been studied in depth is their membrane topologies. Various in silico analyses universally predict that PspB is a bitopic membrane protein with the C terminus inside. However, similar analyses yield conflicting predictions for PspC: a bitopic membrane protein with the C terminus inside, a bitopic membrane protein with the C terminus outside, or a polytopic protein with both termini inside. Previous studies of Escherichia coli PspB-LacZ and PspC-PhoA fusion proteins supported bitopic topologies, with the PspB C terminus inside and the PspC C terminus outside. Here we have used a series of independent approaches to determine the membrane topologies of PspB and PspC in Y. enterocolitica. Our data support the predicted arrangement of PspB, with its C terminus in the cytoplasm. In contrast, data from multiple independent approaches revealed that both termini of PspC are located in the cytoplasm. Additional experiments suggested that the C terminus of PspC might be the recognition site for the FtsH protease and an interaction interface with PspA, both of which would be compatible with its newly proposed cytoplasmic location. This unexpected arrangement of PspC allows a new model for events underlying activation of the Psp response, which is an excellent fit with observations from various previous studies.

Development of a high‐throughput screening approach targeting inhibitors of bacterial cell wall synthesis

Many current antimicrobials act upon the synthesis of the peptidoglycan (PG) cell wall. The proteins that synthesize, assemble, and degrade PG are critical for cell viability. However, evolving antibiotic resistance places a new urgency in prokaryotic biology for new targets. In rod‐shaped bacteria, a large complex of protein interactions coordinate PG synthesis. At the heart of this complex exists an association recently found between two well‐conserved proteins ‐ an actin homolog (MreB) and a bitopic membrane protein (RodZ), both vital for cell wall maintenance. The interaction is confirmed in several species by various methods, one including a bacterial two‐hybrid system. We have adapted this system to high‐throughput screening (HTS) to search for inhibitors of the Caulobacter crescentus MreB‐RodZ interaction. This 384‐ well adaptation involves several original protocol modifications for final assay validation. With this assay, we have screened more than 70,000 unique compounds, producing 485 potential hits. A secondary counter‐screen and confirmatory experiments determine hits specific to RodZ‐MreB interaction. This work demonstrates two‐hybrid HTS assay feasibility and may potentially provide novel inhibitors of these proteins. It also may prove useful for testing of other essential protein interactions. This work is funded by a NIH Exploratory Research Grant.

Quaternary Structure of the Oxaloacetate Decarboxylase Membrane Complex and Mechanistic Relationships to Pyruvate Carboxylases

The oxaloacetate decarboxylase primary Na(+) pump (OAD) is an essential membrane protein complex that functions in the citrate fermentation pathway of some pathogenic bacteria under anaerobic conditions. OAD contains three different subunits: Oad-α, a biotinylated extrinsic protein that catalyzes the α-ketodecarboxylation of oxaloacetate; Oad-γ, a structural bitopic membrane protein whose cytosolic tail (named as Oad-γ') binds tightly to Oad-α; and Oad-β, a multispan transmembrane α-helical protein that constitutes the Na(+) channel. How OAD is organized structurally at the membrane and what the molecular determinants are that lead to an efficient energy coupling mechanism remain elusive. In the present work, we elucidate the stoichiometry of the native complex as well as the low resolution structure of the peripheral components of OAD (Oad-α and Oad-γ') by small angle x-ray scattering. Our results point to a quaternary assembly similar to the pyruvate carboxylase complex organization. Herein, we propose a model in which the association in pairs of Oad-α dimers, mediated by Oad-γ, results in the acquisition of a functional oligomeric state at the bacterial membrane. New structural insights for the conformational rearrangements associated with the carboxylbiotin transfer reaction within OAD are provided.

The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer

Agrobacterium tumefaciens is a plant pathogen that utilizes a type IV secretion system (T4SS) to transfer DNA and effector proteins into host cells. In this study we discovered that an alpha-crystallin type small heat-shock protein (alpha-Hsp), HspL, is a molecular chaperone for VirB8, a T4SS assembly factor. HspL is a typical alpha-Hsp capable of protecting the heat-labile model substrate citrate synthase from thermal aggregation. It forms oligomers in a concentration-dependent manner in vitro. Biochemical fractionation revealed that HspL is mainly localized in the inner membrane and formed large complexes with certain VirB protein subassemblies. Protein-protein interaction studies indicated that HspL interacts with VirB8, a bitopic integral inner membrane protein that is essential for T4SS assembly. Most importantly, HspL is able to prevent the aggregation of VirB8 fused with glutathione S-transferase in vitro, suggesting that it plays a role as VirB8 chaperone. The chaperone activity of two HspL variants with amino acid substitutions (F98A and G118A) for both citrate synthase and glutathione S-transferase-VirB8 was reduced and correlated with HspL functions in T4SS-mediated DNA transfer and virulence. This study directly links in vitro and in vivo functions of an alpha-Hsp and reveals a novel alpha-Hsp function in T4SS stability and bacterial virulence.

Protein localization and dynamics within a bacterial organelle

Protein localization mechanisms dictate the functional and structural specialization of cells. Of the four polar surface organelles featured by the dimorphic bacterium Caulobacter crescentus, the stalk, a cylindrical extension of all cell envelope layers, is the least well characterized at the molecular level. Here we apply a powerful experimental scheme that integrates genetics with high-throughput localization to discover StpX, an uncharacterized bitopic membrane protein that modulates stalk elongation and is sequestered to the stalk. In stalkless mutants StpX is dispersed. Two populations of StpX were discernible within the stalk with different mobilities: an immobile one near the stalk base and a mobile one near the stalk tip. Molecular anatomy provides evidence that (i) the StpX transmembrane domain enables access to the stalk organelle, (ii) the N-terminal periplasmic domain mediates retention in the stalk, and (iii) the C-terminal cytoplasmic domain enhances diffusion within the stalk. Moreover, the accumulation of StpX and an N-terminally truncated isoform is differentially coordinated with the cell cycle. Thus, at the submicron scale the localization and the mobility of a protein are precisely regulated in space and time and are important for the correct organization of a subcellular compartment or organelle such as the stalk.

Bacterial actin MreB assembles in complex with cell shape protein RodZ

Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB-RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm.

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX, DedD, and RlpA) in Escherichia coli Cell Constriction

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an alpha-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain ((E)FtsN) of at most 35 residues that is centered about helix H2 in the periplasm. (E)FtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain ((S)FtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of (S)FtsN depended on the ability of cells to constrict and, thus, on the activity of (E)FtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.

The dimer formed by the periplasmic domain of EpsL from the Type 2 Secretion System of Vibrio parahaemolyticus

The Type 2 Secretion System (T2SS), occurring in many Gram-negative bacteria, is responsible for the transport of a diversity of proteins from the periplasm across the outer membrane into the extracellular space. In Vibrio cholerae, the T2SS secretes several unrelated proteins including the major virulence factor cholera toxin. The T2SS consists of three sub-assemblies, one of which is the Inner Membrane Complex which contains multiple copies of five proteins, including the bitopic membrane protein EpsL. Here, we report the 2.3A resolution crystal structure of the periplasmic domain of EpsL (peri-EpsL) from Vibrio parahaemolyticus, which is 56% identical in sequence to its homolog in V. cholerae. The domain adopts a circular permutation of the "common" ferredoxin fold with two contiguous sub-domains. Remarkably, this infrequently occurring permutation was for the first time observed in the periplasmic domain of EpsM (peri-EpsM), another T2SS protein which interacts with EpsL. These two domains are 18% identical in sequence which may indicate a common evolutionary origin. Both peri-EpsL and peri-EpsM form dimers, but the organization of the subunits in these dimers appears to be entirely different. We have previously shown that the cytoplasmic domain of EpsL is also dimeric and forms a heterotetramer with the first domain of the "secretion ATPase" EpsE. The latter enzyme is most likely hexameric. The possible consequences of the combination of the different symmetries of EpsE and EpsL for the architecture of the T2SS are discussed.

The divisomal protein DivIB contains multiple epitopes that mediate its recruitment to incipient division sites

Bacterial cytokinesis is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. DivIB and its orthologue FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. A molecular dissection approach was used to determine which of these domains are essential for recruitment of DivIB to incipient division sites and for its cell division functions. We show that DivIB has three molecular epitopes that mediate its localization to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and we have used this information to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localization epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be individually non-essential.

Contribution of the FtsQ Transmembrane Segment to Localization to the Cell Division Site

The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.

How important are transmembrane helices of bitopic membrane proteins?

The topology of a bitopic membrane protein consists of a single transmembrane helix connecting two extra-membranous domains. As opposed to helices from polytopic proteins, the transmembrane helices of bitopic proteins were initially considered as merely hydrophobic anchors, while more recent studies have begun to shed light on their role in the protein's function. Herein the overall importance of transmembrane helices from bitopic membrane proteins was analyzed using a relative conservation analysis. Interestingly, the transmembrane domains of bitopic proteins are on average, significantly more conserved than the remainder of the protein, even when taking into account their smaller amino acid repertoire. Analysis of highly conserved transmembrane domains did not reveal any unifying consensus, pointing to a great diversity in their conservation patterns. However, Fourier power spectrum analysis was able to show that regardless of the conservation motif, in most sequences a significant conservation moment was observed, in that one side of the helix was conserved while the other was not. Taken together, it may be possible to conclude that a significant proportion of transmembrane helices from bitopic membrane proteins participate in specific interactions, in a variety of modes in the plane of the lipid bilayer.

Dimeric structure of the cell shape protein MreC and its functional implications

The bacterial actin homologue MreB forms helical filaments in the cytoplasm of rod-shaped bacteria where it helps maintain the shape of the cell. MreB is co-transcribed with mreC that encodes a bitopic membrane protein with a major periplasmic domain. Like MreB, MreC is localized in a helical pattern and might be involved in the spatial organization of the peptidoglycan synthesis machinery. Here, we present the structure of the major, periplasmic part of MreC from Listeria monocytogenes at 2.5 A resolution. MreC forms a dimer through an intimate contact along an N-terminal alpha-helix that connects the transmembrane region with two C-terminal beta-domains. The translational relationship between the molecules enables, in principle, filament formation. One of the beta-domains shows structural similarity to the chymotrypsin family of proteins and possesses a highly conserved Thr Ser dipeptide. Unexpectedly, mutagenesis studies show that the dipeptide is dispensable for maintaining cell shape and viability in both Escherichia coil and Bacillus subtilis. Bacterial two-hybrid experiments reveal that MreC Interacts with high-molecular-weight penicillin-binding proteins (PBPs), rather than with low-molecular-weight endo- and carboxypeptidases, indicating that MreC might act as a scaffold to which the murein synthases are recruited in order to spatially organize the synthesis of new cell wall material. Deletion analyses indicate which domains of B. subtilis MreC are required for interaction with MreD as well as with the PBPs.

Topologically Fixed SecG Is Fully Functional

It has been proposed that the bitopic membrane protein SecG undergoes topology inversion during translocation of (pre)proteins via SecYEG. Here we show that SecG covalently cross-linked to SecY cannot invert its topology while remaining fully functional in protein translocation. Our results strongly disfavor topology inversion of SecG during protein translocation.

Putative membrane assembly of EtpM-colicin V chimeras

EtpM of the enterohemorrhagic E. coli O157:H7 is a bitopic membrane protein of the type II protein secretion apparatus. There is a twin-arginine (RR) motif in front of its signal anchor, suggesting a Tat-dependent membrane targeting of EtpM. By exploiting the periplasmic bactericidal activity of colicin V (ColV), we constructed EtpM-ColV fusions and studied the EtpM-mediated translocation of ColV. The wild type strain and the Δ tatC mutant were killed by the expressed fusions and were fully protected from the killing effect by the ColV-specific immunity protein. In contrast, cold-inactivation of YidC, which is generally required for integral membrane protein assembly, significantly attenuated the killing effect in the cold-sensitive yidC mutant. These results confirmed the predicted N(in)-C(out) EtpM topology, and suggests an EtpM-mediated, Tat-independent and YidC-dependent translocation of ColV.

Topology and membrane interaction of Helicobacter pylori ComB proteins involved in natural transformation competence

AbstractThe human gastric pathogen Helicobacter pylori is naturally competent for genetic transformation. The H. pylori comB gene cluster encodes the VirB4-homologous ATPase ComB4 and the structural proteins ComB7 – ComB10, which share significant sequence identity to the Agrobacterium tumefaciens virB-encoded type IV secretion system. To study the topology of the ComB7 – 10 proteins, we applied TnMax transposon mutagenesis by generating fusions of ComB proteins with mature β-lactamase (BlaM) or alkaline phosphatase (PhoA). Our data show that the putative lipoprotein ComB7 is secreted and is found membrane-attached, probably by its lipid anchor. According to our topology mapping ComB8 is a bitopic membrane protein with a short N-terminal portion in the cytoplasm and the remainder of the protein expanding into the periplasmic space. ComB9 was verified as a periplasmic protein, tightly attached to the membrane. The N-terminus of ComB10 is anchored in the cytoplasmic membrane and the major portion of the protein, including a putative coiled-coil domain, is located in the periplasm. Limited protease digestion and protein extraction under different salt and pH conditions confirmed the periplasmic localization and the tight membrane association of the ComB protein complex. A hypothetical model of the ComB DNA transformation pore in H. pylori is presented.

Analysis of ftsQ mutant alleles in Escherichia coli: complementation, septal localization, and recruitment of downstream cell division proteins.

FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

The Bacillus subtilis cell division protein FtsL localizes to sites of septation and interacts with DivIC.

FtsL is a small bitopic membrane protein required for vegetative cell division and sporulation in Bacillus subtilis. We investigated its localization by fluorescence microscopy using a green fluorescent protein (GFP) fusion. GFP-FtsL was localized at mid-cell in vegetative cells and at the asymmetric septum in sporulating cells. We also show that FtsL forms a ring-like structure at the division site and that it remains localized at mid-cell during the whole septation process. By yeast two-hybrid analysis and non-denaturing polyacrylamide gel electrophoresis (PAGE) with purified proteins, FtsL was found to interact with another membrane-bound division protein, the FtsL-like DivIC protein.

Cell division in Escherichia coli: role of FtsL domains in septal localization, function, and oligomerization.

In Escherichia coli, nine essential cell division proteins are known to localize to the division septum. FtsL is a 13-kDa bitopic membrane protein with a short cytoplasmic N-terminal domain, a membrane-spanning segment, and a periplasmic domain that has a repeated heptad motif characteristic of leucine zippers. Here, we identify the requirements for FtsL septal localization and function. We used green fluorescent protein fusions to FtsL proteins where domains of FtsL had been exchanged with analogous domains from either its Haemophilus influenzae homologue or the unrelated MalF protein to show that both the membrane-spanning segment and the periplasmic domain of FtsL are required for localization to the division site. Mutagenesis of the periplasmic heptad repeat motif severely impaired both localization and function as well as the ability of FtsL to drive the formation of sodium dodecyl sulfate-resistant multimers in vitro. These results are consistent with the predicted propensity of the FtsL periplasmic domain to adopt a coiled-coiled structure. This coiled-coil motif is conserved in all gram-negative and gram-positive FtsL homologues identified so far. Our data suggest that most of the FtsL molecule is a helical coiled coil involved in FtsL multimerization.

Septal localization of FtsQ, an essential cell division protein in Escherichia coli.

Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.

ComEA is a DNA receptor for transformation of competent Bacillus subtilis.

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.