Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins ...(FliPQR in flagella, SctRST in virulence systems). A fourth essential T3SS protein (FlhB/SctU) functions to "switch" secretion substrate specificity once the growing hook/needle reach their determined length. Here, we present the cryo-electron microscopy structure of an export gate containing the switch protein from a Vibrio flagellar system at 3.2 Å resolution. The structure reveals that FlhB/SctU extends the helical export gate with its four predicted transmembrane helices wrapped around FliPQR/SctRST. The unusual topology of the FlhB/SctU helices creates a loop wrapped around the bottom of the closed export gate. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the T3SS trigger opening of the gate through interactions between FlhB/SctU and FliPQR/SctRST.
Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are ...suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.
Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which ...secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhA.sub.L /SctV.sub.L, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.
The bacterial flagellum is a complex self-assembling nanomachine that confers motility to the cell. Despite great variation across species, all flagella are ultimately constructed from a helical ...propeller that is attached to a motor embedded in the inner membrane. The motor consists of a series of stator units surrounding a central rotor made up of two ring complexes, the MS-ring and the C-ring. Despite many studies, high-resolution structural information is still lacking for the MS-ring of the rotor, and proposed mismatches in stoichiometry between the two rings have long provided a source of confusion for the field. Here, we present structures of the Salmonella MS-ring, revealing a high level of variation in inter- and intrachain symmetry that provides a structural explanation for the ability of the MS-ring to function as a complex and elegant interface between the two main functions of the flagellum-protein secretion and rotation.
Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate ...complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the
virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex.
Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled.
Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, ...the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.
One of the most widespread bacterial secretion systems is the type three secretion system (T3SS), which is critical for the virulence of many pathogens and responsible for assembly of the bacterial ...flagellum. The virulence-associated T3SS is a molecular syringe that uses a highly conserved export apparatus (EA) to secrete effector proteins through a needle directly into the host cell cytoplasm. The flagellar T3SS secretes flagellar subunits through the EA which then self-assemble into a filament. Despite recent progress in the structural biology of T3SS, the highly conserved membrane domains of the five inner membrane proteins of the EA have evaded structural characterisation. Here, multiple cryo-EM structures of complexes of the three core EA proteins from both flagella (FliPQR) and injectisomes (SctRST) are presented, revealing a pseudohexameric, helical complex. Remarkably, the complex is made up of a series of long, highly kinked helical hairpins rather than the predicted canonical transmembrane helices. Many of the hydrophobic residues predicted to lie in transmembrane helices are instead buried at the interfaces between subunits, leaving only a small membrane domain. In the assembled T3SS this complex is encased in the basal body, above the plane of the inner membrane, and connects to the helical filament, whose helical parameters it matches. The membrane domain of the fourth export apparatus protein, FlhB/SctU, is shown to form a complex with FliPQR, and the complex structure suggests a mechanism of how interactions between FlhB and FliPQR could lead to opening of the complex. Finally, stable rings of full-length FlhA/SctV, the last EA protein, were prepared and cryo-EM confirmed the structure of the nonameric cytoplasmic domain. While little high-resolution information of the membrane domain was obtained, these studies provide a framework for future studies. Together these results reveal the structure of part of the secretion pathway and suggest that the core of the export apparatus templates the assembly of the helical filament.
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