Bitopic Membrane Protein Research Articles

Synthetic Assembly of Bacterial Divisome Machinery in Vitro

Bacterial cells grow and reproduce through coordination of many proteins collectively known as the cell division machinery. This machinery facilitates the division and physical separation of one cell into two identical daughter cells. The components of this cell division machinery are similar between bacterial taxa. In Escherichia coli, the cytoskeletal protein FtsZ promotes the formation of a division septum at the cell center and assembles into a large protein structure called the Z‐ring. FtsZ protomers in the Z‐ring form large linear polymers that assemble in a head to tail arrangement. Protein interactions between FtsZ in the Z‐ring and additional cell division proteins direct peptidoglycan synthesis and facilitate septation of the cell. FtsN, which may trigger septation, is a bitopic membrane protein with a small cytoplasmic domain and large periplasmic domain containing a peptidoglycan‐binding SPOR domain. FtsN interacts with other cell division proteins on both sides of the cytoplasmic membrane. In the cytoplasm, FtsN interacts with FtsA, an actin homolog, which tethers FtsZ polymers to the membrane. In the periplasm FtsN interacts with the FtsQBL complex and may help to recruit peptidoglycan synthetases such as FtsI. One proposed linear mechanism for activating cell wall synthesis begins with FtsA, which binds to FtsN in the cytoplasm, and causes FtsN to relay information to proteins in the periplasm, such as FtsQBL, to begin remodeling of the cell wall. Thus, it is critical that FtsN localizes to the future site of septation in order to carry out this function. Here we use purified proteins, including FtsZ, FtsA, and FtsN, to reconstitute the membrane‐spanning division complex in vitro and assemble a synthetic divisome scaffold. This reconstituted divisome subassembly allows us to probe direct protein interactions, protein conformations, assembly architectures, and nucleotide requirements in the cell division pathway, thus providing novel insight into the mechanics of division.

Localization of two interacting protein components of the conjugation machinery of Bacillus subtilis

Conjugation, or mating, is a major form of horizontal gene transfer, contributing to the spread of antibiotic resistance and virulence genes in bacterial populations. During conjugation, DNA is transferred from donor to recipient cell through a large multi-protein DNA translocation channel produced in the donor cell. We are interested in understanding the localization of the conjugation machinery of the conjugative element ICEBs1 of Bacillus subtilis. We previously identified several proteins that make up the ICEBs1 DNA translocation channel, including the peripheral membrane protein ConE and the transmembrane protein ConB. ConE fused to green fluorescent protein (GFP) localizes to the cell membrane, predominantly at the poles. ConE interacts with the bitopic membrane protein ConB. This interaction is functionally important as ConE-GFP mislocalizes to the cytoplasm in a strain deleted for conB. Here, we discovered that a fusion of ConB to the red fluorescent protein mCHERRY also localizes to the cell membrane at the poles, similarly to ConE-GFP. We also show that ConB-mCHERRY localization is independent of other conjugation proteins, but dependent on its own transmembrane segment. Finally, we discovered which domains of ConB are required for ConE-GFP localization. Our work provides new insights into the subcellular localization of two conserved interacting proteins that are critical for mediating horizontal gene transfer.

Soluble TatA forms oligomers that interact with membranes: Structure and insertion studies of a versatile protein transporter

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer.

Fluorescence Localization of the Conjugation Machinery of Bacillus subtilis

Conjugation, or mating, is the transfer of DNA from a donor to a recipient cell through conjugation machinery. Conjugation contributes to bacterial genome innovation and the spread of virulence and antibiotic resistance genes between bacteria. DNA is transferred during conjugation through a multi‐protein DNA translocation channel, classified as a Type IV secretion system (T4SS), embedded in the membrane. Our work focuses on characterizing the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Previously, we identified several protein components of the ICEBs1 conjugation machinery, including the bitopic membrane protein ConB and the peripheral membrane ATPase ConE. We have used fluorescence microscopy to observe the locations of ConB and ConE inside cells. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane, predominantly at the cell poles. Here, we show that a fusion of ConB to the red fluorescent protein mCHERRY localizes similarly to ConE‐GFP. Consistent with ConE lacking predicted transmembrane segments, ConE‐GFP’s localization to the membrane requires ConB. In contrast, localization of ConB‐mCHERRY to the membrane does not depend on other conjugation proteins. Current studies are focused on which domains of ConB are required for its own localization as well as the localization of ConE.Support or Funding InformationThis research was funded by Suffolk University and an NSF‐RUI grant to M. Berkmen.

The case for class II bacteriocins: A biophysical approach using “suicide probes” in receptor-free hosts to study their mechanism of action

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called “suicide probes”. The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.

Multi-functional regulator MapZ controls both positioning and timing of FtsZ polymerization.

The tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization.

A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum.

Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP–FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.

New insights into enterocin CRL35: mechanism of action and immunity revealed by heterologous expression in Escherichia coli.

The role of the class IIa bacteriocin membrane receptor protein remains unclear, and the following two different mechanisms have been proposed: the bacteriocin could interact with the receptor changing it to an open conformation or the receptor might act as an anchor allowing subsequent bacteriocin insertion and membrane disruption. Bacteriocin-producing cells synthesize an immunity protein that forms an inactive bacteriocin-receptor-immunity complex. To better understand the molecular mechanism of enterocin CRL35, the peptide was expressed as the suicidal probe EtpM-enterocin CRL35 in Escherichia coli, a naturally insensitive microorganism since it does not express the receptor. When the bacteriocin is anchored to the periplasmic face of the plasma membrane through the bitopic membrane protein, EtpM, E. coli cells depolarize and die. Moreover, co-expression of the immunity protein prevents the deleterious effect of EtpM-enterocin CRL35. The binding and anchoring of the bacteriocin to the membrane has demonstrated to be a sufficient condition for its membrane insertion. The final step of membrane disruption by EtpM-enterocin CRL35 is independent from the receptor, which means that the mannose PTS might not be involved in the pore structure. In addition, the immunity protein can protect even in the absence of the receptor.

Decoupled side chain and backbone dynamics for proton translocation - M2 of influenza A.

The 97 amino acid bitopic membrane protein M2 of influenza A forms a tetrameric bundle in which two of the monomers are covalently linked via a cysteine bridge. In its tetrameric assembly the protein conducts protons across the viral envelope and within intracellular compartments during the infectivity cycle of the virus. A key residue in the translocation of the protons is His-37 which forms a planar tetrad in the configuration of the bundle accepting and translocating the incoming protons from the N terminal side, exterior of the virus, to the C terminal side, inside the virus. With experimentally available data from NMR spectroscopy of the transmembrane domains of the tetrameric M2 bundle classical MD simulations are conducted with the protein bundle in different protonation stages in respect to His-37. A full correlation analysis (FCA) of the data sets with the His-37 tetrad either in a fully four times unprotonated or protonated state, assumed to mimic high and low pH in vivo, respectively, in both cases reveal asymmetric backbone dynamics. His-37 side chain rotation dynamics is increased at full protonation of the tetrad compared to the dynamics in the fully unprotonated state. The data suggest that proton translocation can be achieved by decoupled side chain or backbone dynamics. Graphical abstract Visualization of the tetrameric bundle of the transmembrane domains of M2 of influenza A after 200 ns of MD simulations (upper left). The four histidine residues 37 are either not protonated as in M20 or fully protonated is in M24+. The asymmetric dynamics of the backbones are shown after a full correlation analysis (FCA) in blue (lower left). The rotational dynamics of the χ2 dihedral angles of the histidines in M20 (upper right) are less than those in M24+ (lower right).

Structural Insights into Binding of the Antifungal Drug Fluconazole to Saccharomyces cerevisiae Lanosterol 14α-Demethylase.

Infections by fungal pathogens such as Candida albicans and Aspergillus fumigatus and their resistance to triazole drugs are major concerns. Fungal lanosterol 14α-demethylase belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes. This monospanning bitopic membrane protein is involved in ergosterol biosynthesis and is the primary target of azole antifungal drugs, including fluconazole. The lack of high-resolution structural information for this drug target from fungal pathogens has been a limiting factor for the design of modified triazole drugs that will overcome resistance. Here we report the X-ray structure of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase in complex with fluconazole at a resolution of 2.05 Å. This structure shows the key interactions involved in fluconazole binding and provides insight into resistance mechanisms by revealing a water-mediated hydrogen bonding network between the drug and tyrosine 140, a residue frequently found mutated to histidine or phenylalanine in resistant clinical isolates.

Roles for both FtsA and the FtsBLQ subcomplex in FtsN-stimulated cell constriction in Escherichia coli.

Escherichia coli FtsN is a bitopic membrane protein that is essential for triggering active cell constriction. A small periplasmic subdomain ((E) FtsN) is required and sufficient for function, but its mechanism of action is unclear. We isolated extragenic (E) FtsN*-suppressing mutations that restore division in cells producing otherwise non-functional variants of FtsN. These mapped to the IC domain of FtsA in the cytoplasm and to small subdomains of the FtsB and FtsL proteins in the periplasm. All FtsB and FtsL variants allowed survival without (E) FtsN, but many then imposed a new requirement for interaction between the cytoplasmic domain of FtsN ((N) FtsN) and FtsA. Alternatively, variants of FtsA, FtsB or FtsL acted synergistically to allow cell division in the complete absence of FtsN. Strikingly, moreover, substitution of a single residue in FtsB (E56) proved sufficient to rescue ΔftsN cells as well. In FtsN(+) cells, (E) FtsN*-suppressing mutations promoted cell fission at an abnormally small cell size, and caused cell shape and integrity defects under certain conditions. This and additional evidence support a model in which FtsN acts on either side of the membrane to induce a conformational switch in both FtsA and the FtsBLQ subcomplex to de-repress septal peptidoglycan synthesis and membrane invagination.

Structure based computational assessment of channel properties of assembled ORF-8a from SARS-CoV.

ABSTRACTORF 8a is a short 39 amino acid bitopic membrane protein encoded by severe acute respiratory syndrome causing corona virus (SARS‐CoV). It has been identified to increase permeability of the lipid membrane for cations. Permeability is suggested to occur due to the assembly of helical bundles. Computational models of a pentameric assembly of 8a peptides are generated using the first 22 amino acids, which include the transmembrane domain. Low energy structures reveal a hydrophilic pore mantled by residues Thr‐8, and −18, Ser‐11, Cys‐13, and Arg‐22. Potential of mean force (PMF) profiles for mono (Na+, K+, Cl−) and divalent (Ca2+) ions along the pore are calculated. The data support experimental findings of a weak cation selectivity of the channel. Calculations on 8a are compared to data derived for a pentameric bundle consisting of the M2 helices of the bacterial pentameric ligand gated ion channel GLIC (3EHZ). PMF curves of both, bundles 8a and M2, show sigmoidal shaped profiles. In comparison to the data for the M2‐GLIC model, data of the 8a bundle show lower amplitude of the PMF values between maximum and minimum and less discrimination amongst ions. Proteins 2015; 83:300–308. © 2014 Wiley Periodicals, Inc.

A role for FtsA in SPOR-independent localization of the essential Escherichia coli cell division protein FtsN.

FtsN is a bitopic membrane protein and the last essential component to localize to the Escherichia coli cell division machinery, or divisome. The periplasmic SPOR domain of FtsN was previously shown to localize to the divisome in a self-enhancing manner, relying on the essential activity of FtsN and the peptidoglycan synthesis and degradation activities of FtsI and amidases respectively. Because FtsN has a known role in recruiting amidases and is predicted to stimulate the activity of FtsI, it follows that FtsN initially localizes to division sites in a SPOR-independent manner. Here, we show that the cytoplasmic and transmembrane domains of FtsN (FtsN(Cyto - TM)) facilitated localization of FtsN independently of its SPOR domain but dependent on the early cell division protein FtsA. In addition, SPOR-independent localization preceded SPOR-dependent localization, providing a mechanism for the initial localization of FtsN. In support of the role of FtsNCyto - TM in FtsN function, a variant of FtsN lacking the cytoplasmic domain localized to the divisome but failed to complement an ftsN deletion unless it was overproduced. Simultaneous removal of the cytoplasmic and SPOR domains abolished localization and complementation. These data support a model in which FtsA-FtsN interaction recruits FtsN to the divisome, where it can then stimulate the peptidoglycan remodelling activities required for SPOR-dependent localization.

Self-Assembly of the Viral Channel Forming Protein Vpu of Hiv-1 using Coarse-Graining Molecular Dynamics Simulations

Vpu of human immunodeficiency virus type 1 (HIV-1) which is an 81 amino acid bitopic membrane protein known to homo-oligomerize. It is known for its ability to form channels and to assemble into host factors. The assembly process and the oligomer state of Vpu are not known. Coarse-graining molecular dynamics simulations are performed with full-length Vpu in lipid bilayers. Full-length Vpu is constructed by linking an experimentally derived structural model of the cytoplasm domain with a computationally generated transmembrane domain (TMD). Simulation boxes are generated harboring selected numbers of VPUs. The simulations identify that Vpu assembles via several binding sites into a series of oligomeric states. Most striking, in a simulation of four Vpu's the proteins form a symmetric bundle which is a close match to known channel architectures. The tetramer is followed a dimer formation. Not only TMDs but also cytoplasm domains are involved in the dynamics of self-assembly.

Qualitative computational bioanalytics: Assembly of viral channel-forming peptides around mono and divalent ions

A fine-grained docking protocol was used to generate a bundle-like structure of the bitopic membrane protein Vpu from HIV-1. Vpu is a type I membrane protein with 81 amino acids. It is proposed that Vpu forms ion- and substrate-conducting bundles, which are located at the plasma membrane in the infected cell. The Vpu1–32 peptide that includes the transmembrane domain (TMD) is assembled into homo-pentameric bundles around prepositioned Na, K, Ca or Cl ions. For bundles with the lowest energy, the TMDs generate a hydrophobic pore. Bundles in which Ser-24 faces the pore have higher energy. The tilt of the helices in the lowest energy bundles is larger than bundles with serines facing the pore. Left-handed bundles are lowest in energy where the ions are located at the serines.

Assembling viral channel forming proteins: Vpu from HIV‐1

Different routes of assembly are probed for the transmembrane domain (TMD) of the bitopic membrane protein Vpu from HIV-1. Vpu is responsible for the amplification of viral release from the host cell. The mode of action includes (i) heteroassembly with host factors and (ii) the formation of homo-oligomers, which are able to conduct ions across the lipid membrane. Two different routes of assembling short sequences of the N terminus, including the TMD of Vpu, Vpu1-32, and Vpu8-26, are presented by using a combination of classical molecular dynamics (MD) simulations combined with a docking approach. The rim of alanines (Ala-8, -11, -15, and -19) resembles an interlocking motif for the sequential assembly into a dimer and trimer. Simultaneous assembly results in oligomeric bundles (trimers to pentamers) with either tryptophans (Trp-23) or purely hydrophobic residues facing the center. Bundles, with serines facing the pore (Ser-24), are energetically not the lowest structures. For pentameric bundles with Ser-24 facing the pore, no water column develops during a short 25 ns MD simulation.

DNA substrate-induced activation of the Agrobacterium VirB/VirD4 type IV secretion system.

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.

Enough is enough: TatA demand during Tat-dependent protein transport

The twin-arginine translocation (Tat1) pathway is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. In chloroplasts and Gram-negative bacteria, Tat translocase consists of the integral subunits TatB and TatC, which are assumed to constitute the membrane receptor, and TatA, a bitopic membrane protein being responsible in a yet unknown manner for the membrane translocation step. Antibody inhibition of intrinsic thylakoidal TatA activity and recovery of transport by heterologously expressed, purified TatA allowed to exactly quantify the amount of TatA required to catalyse membrane transport of the model Tat substrate 16/23. We can show that TatA concentrations in the 100nM range are sufficient to efficiently catalyse membrane transport of the protein, which corresponds well to the amount of TatA identified in thylakoids. Furthermore, TatA shows cooperativity in its catalytic activity suggesting that Tat translocase operates as an allosteric enzyme complex.

A Specific Role for the ZipA Protein in Cell Division: STABILIZATION OF THE FtsZ PROTEIN

In Escherichia coli, the cell division protein FtsZ is anchored to the cytoplasmic membrane by the action of the bitopic membrane protein ZipA and the cytoplasmic protein FtsA. Although the presence of both ZipA and FtsA is strictly indispensable for cell division, an FtsA gain-of-function mutant FtsA* (R286W) can bypass the ZipA requirement for cell division. This observation casts doubts on the role of ZipA and its need for cell division. Maxicells are nucleoid-free bacterial cells used as a whole cell in vitro system to probe protein-protein interactions without the need of protein purification. We show that ZipA protects FtsZ from the ClpXP-directed degradation observed in E. coli maxicells and that ZipA-stabilized FtsZ forms membrane-attached spiral-like structures in the bacterial cytoplasm. The overproduction of the FtsZ-binding ZipA domain is sufficient to protect FtsZ from degradation, whereas other C-terminal ZipA partial deletions lacking it are not. Individual overproduction of the proto-ring component FtsA or its gain-of-function mutant FtsA* does not result in FtsZ protection. Overproduction of FtsA or FtsA* together with ZipA does not interfere with the FtsZ protection. Moreover, neither FtsA nor FtsA* protects FtsZ when overproduced together with ZipA mutants lacking the FZB domain. We propose that ZipA protects FtsZ from degradation by ClpP by making the FtsZ site of interaction unavailable to the ClpX moiety of the ClpXP protease. This role cannot be replaced by either FtsA or FtsA*, suggesting a unique function for ZipA in proto-ring stability.

Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment

The Type VII protein translocation/secretion system, unique to Gram-positive bacteria, is a key virulence determinant in Staphylococcus aureus. We aim to characterize the architecture of this secretion machinery and now describe the present study of S. aureus EssB, a 52 kDa bitopic membrane protein essential for secretion of the ESAT-6 (early secretory antigenic target of 6 kDa) family of proteins, the prototypic substrate of Type VII secretion. Full-length EssB was heterologously expressed in Escherichia coli, solubilized from the bacterial membrane, purified to homogeneity and shown to be dimeric. A C-terminal truncation, EssB∆C, and two soluble fragments termed EssB-N and EssB-C, predicted to occur on either side of the cytoplasmic membrane, have been successfully purified in a recombinant form, characterized and, together with the full-length protein, used in crystallization trials. EssB-N, the 25 kDa N-terminal cytoplasmic fragment, gave well-ordered crystals and we report the structure, determined by SAD (single-wavelength anomalous diffraction) targeting an SeMet (selenomethionine) derivative, refined to atomic (1.05 Å; 1 Å=0.1 nm) resolution. EssB-N is dimeric in solution, but crystallizes as a monomer and displays a fold comprised of two globular domains separated by a cleft. The structure is related to that of serine/threonine protein kinases and the present study identifies that the Type VII secretion system exploits and re-uses a stable modular entity and fold that has evolved to participate in protein–protein interactions in a similar fashion to the catalytically inert pseudokinases.