Cryo-EM structure of a type IV secretion system.

Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.

Design, synthesis, and evaluation of peptide-imidazo[1,2-a]pyrazine bioconjugates as potential bivalent inhibitors of the VirB11 ATPase HP0525.

Helicobacter pylori (H. pylori) infections have been implicated in the development of gastric ulcers and various cancers: however, the success of current therapies is compromised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular nanomachine that transfers toxic bacterial factors and plasmid DNA between bacterial cells, thus contributing to the spread of antibiotic resistance. A key component of the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We have previously reported the design and synthesis of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their selectivity, and potentially develop these compounds as tools for probing the assembly of the HP0525 hexamer, we have explored the design and synthesis of potential bivalent inhibitors. We used the structural details of the subunit-subunit interactions within the HP0525 hexamer to design peptide recognition moieties of the subunit interface. Different methods (cross metathesis, click chemistry, and cysteine-malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were explored, as well as peptides spanning larger or smaller regions of the interface. The IC50 values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and the bivalent inhibitors, were related to docking studies with the HP0525 crystal structure and to molecular dynamics simulations of the peptide recognition moieties.

The Remarkable Biomechanical Properties of the Type 1 Chaperone-Usher Pilus: A Structural and Molecular Perspective.

Chaperone-usher (CU) pili are long, supramolecular protein fibers tethered to the surface of numerous bacterial pathogens. These virulence factors function primarily in bacterial adhesion to host tissues, but they also mediate biofilm formation. Type 1 and P pili of uropathogenic Escherichia coli (UPEC) are the two best-studied CU pilus examples, and here we primarily focus on the former. UPEC can be transmitted to the urinary tract by fecal shedding. It can then ascend up the urinary tract and cause disease by invading and colonizing host tissues of the bladder, causing cystitis, and the kidneys, causing pyelonephritis. FimH is the subunit displayed at the tip of type 1 pili and mediates adhesion to mannosylated host cells via a unique catch-bond mechanism. In response to shear forces caused by urine flow, FimH can transition from a low-affinity to high-affinity binding mode. This clever allosteric mechanism allows UPEC cells to remain tightly attached during periods of urine flow, while loosening their grip to allow dissemination through the urinary tract during urine stasis. Moreover, the bulk of a CU pilus is made up of the rod, which can reversibly uncoil in response to urine flow to evenly spread the tensile forces over the entire pilus length. We here explore the novel structural and mechanistic findings relating to the type 1 pilus FimH catch-bond and rod uncoiling and explain how they function together to enable successful attachment, spread, and persistence in the hostile urinary tract.

X-ray crystal structures of the type IVb secretion system DotB ATPases.

Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilizes a Type IVb secretion (T4bS) system termed "dot/icm" to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS-like N-terminal domain coupled to a RecA-like C-terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and ß, with an αßαßαß configuration. The existence of α and ß conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααßααß configuration. The two conformers co-exist regardless of the nucleotide-bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug design to develop more specific antibiotics to tackle Legionnaire's disease. PDB Code(s): Will; be; provided.

Crystal structure of the Legionella pneumophila Lpg2936 in complex with the cofactor S-adenosyl-L-methionine reveals novel insights into the mechanism of RsmE family methyltransferases.

The methylation of U1498 located in the 16S ribosomal RNA of Escherichia coli is an important modification affecting ribosomal activity. RsmE methyltransferases methylate specifically this position in a mechanism that requires an S-adenosyl-L-methionine (AdoMet) molecule as cofactor. Here we report the structure of Apo and AdoMet-bound Lpg2936 from Legionella pneumophila at 1.5 and 2.3 Å, respectively. The protein comprises an N-terminal PUA domain and a C-terminal SPOUT domain. The latter is responsible for protein dimerization and cofactor binding. Comparison with similar structures suggests that Lpg2936 is an RsmE-like enzyme that can target the equivalent of U1498 in the L. pneumophila ribosomal RNA, thereby potentially enhancing ribosomal activity during infection-mediated effector production. The multiple copies of the enzyme found in both structures reveal a flexible conformation of the bound AdoMet ligand. Isothermal titration calorimetry measurements suggest an asymmetric two site binding mode. Our results therefore also provide unprecedented insights into AdoMet/RsmE interaction, furthering our understanding of the RsmE catalytic mechanism.

Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane ß-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.

Recent advances in the structural and molecular biology of type IV secretion systems.

Bacteria use type IV secretion (T4S) systems to deliver DNA and protein substrates to a diverse range of prokaryotic and eukaryotic target cells. T4S systems have great impact on human health, as they are a major source of antibiotic resistance spread among bacteria and are central to infection processes of many pathogens. Therefore, deciphering the structure and underlying translocation mechanism of T4S systems is crucial to facilitate development of new drugs. The last five years have witnessed considerable progress in unraveling the structure of T4S system subassemblies, notably that of the T4S system core complex, a large 1 MegaDalton (MDa) structure embedded in the double membrane of Gram-negative bacteria and made of 3 of the 12 T4S system components. However, the recent determination of the structure of -3MDa assembly of 8 of these components has revolutionized our views of T4S system architecture and opened up new avenues of research, which are discussed in this review.

Structure of a type IV secretion system.

Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems.

Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization.

OBJECTIVES: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). METHODS: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. RESULTS: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. CONCLUSIONS: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens.

Molecular basis of usher pore gating in Escherichia coli pilus biogenesis.

Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane ß-barrel translocation domain (TD), a ß-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two C-terminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (ß12-13 loop) was found to facilitate pore opening. Mutation of the ß12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.

Structure of the VirB4 ATPase, alone and bound to the core complex of a type IV secretion system.

Type IV secretion (T4S) systems mediate the transfer of proteins and DNA across the cell envelope of bacteria. These systems play important roles in bacterial pathogenesis and in horizontal transfer of antibiotic resistance. The VirB4 ATPase of the T4S system is essential for both the assembly of the system and substrate transfer. In this article, we present the crystal structure of the C-terminal domain of Thermoanaerobacter pseudethanolicus VirB4. This structure is strikingly similar to that of another T4S ATPase, VirD4, a protein that shares only 12% sequence identity with VirB4. The VirB4 domain purifies as a monomer, but the full-length protein is observed in a monomer-dimer equilibrium, even in the presence of nucleotides and DNAs. We also report the negative stain electron microscopy structure of the core complex of the T4S system of the Escherichia coli pKM101 plasmid, with VirB4 bound. In this structure, VirB4 is also monomeric and bound through its N-terminal domain to the core's VirB9 protein. Remarkably, VirB4 is observed bound to the side of the complex where it is ideally placed to play its known regulatory role in substrate transfer.

Bromopyridazinedione-mediated protein and peptide bioconjugation.

Bromopyridazinedione-mediated bioconjugation to a cysteine containing protein and a disulfide containing peptide is described. The conjugates are cleavable in an excess of thiol, including cytoplasmically-relevant concentrations of glutathione, and show a high level of hydrolytic stability. The constructs have the potential for four points of chemical attachment.

Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding.

Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3'-to-5' directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a "closed" state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an "open" state that differs by an ∼160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.

Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis.

Controlling maleimide hydrolysis allows the modular construction of bromomaleimide-mediated bioconjugates which are either stable or cleavable in an aqueous, thiol-mediated reducing environment. The application of this methodology to reversible protein biotinylation, the irreversible labeling of peptide disulfide bonds and the assembly of stable, fluorescein-labelled glycoprotein mimics is described.

An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect.

Agrobacterium VirB7, VirB9, and VirB10 form a "core complex" during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra(+)) but did block pilus production (Pil(-)). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra(+) Pil(-) phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.

Structural insights into the membrane-extracted dimeric form of the ATPase TraB from the Escherichia coli pKM101 conjugation system.

BACKGROUND: Type IV secretion (T4S) systems are involved in secretion of virulence factors such as toxins or transforming molecules, or bacterial conjugation. T4S systems are composed of 12 proteins named VirB1-B11 and VirD4. Among them, three ATPases are involved in the assembly of the T4S system and/or provide energy for substrate transfer, VirB4, VirB11 and VirD4. The X-ray crystal structures of VirB11 and VirD4 have already been solved but VirB4 has proven to be reluctant to any structural investigation so far. RESULTS: Here, we have used small-angle X-ray scattering to obtain the first structural models for the membrane-extracted, dimeric form of the TraB protein, the VirB4 homolog encoded by the E. coli pKM101 plasmid, and for the monomeric soluble form of the LvhB4 protein, the VirB4 homolog of the T4S system encoded by the Legionella pneumophila lvh operon. We have obtained the low resolution structures of the full-length TraB and of its N- and C-terminal halves. From these SAXS models, we derive the internal organisation of TraB. We also show that the two TraB N- and C-terminal domains are independently involved in the dimerisation of the full-length protein. CONCLUSIONS: These models provide the first structural insights into the architecture of VirB4 proteins. In particular, our results highlight the modular arrangement and functional relevance of the dimeric-membrane-bound form of TraB.

Biochemical dissection of the ATPase TraB, the VirB4 homologue of the Escherichia coli pKM101 conjugation machinery.

Type IV secretion (T4S) systems are involved in several secretion processes, including secretion of virulence factors, such as toxins or transforming molecules, or bacterial conjugation whereby two mating bacteria exchange genetic material. T4S systems are generally composed of 12 protein components, three of which, termed VirB4, VirB11, and VirD4, are ATPases. VirB4 is the largest protein of the T4S system, is known to play a central role, and interacts with many other T4S system proteins. In this study, we have biochemically characterized the protein TraB, a VirB4 homologue from the pKM101 conjugation T4S system. We demonstrated that TraB is a modular protein, composed of two domains, both able to bind DNA in a non-sequence-specific manner. Surprisingly, both TraB N- and C-terminal domains can bind ATP, revealing a new degenerated nucleotide-binding site in the TraB N-terminal domain. TraB purified from the membrane forms stable dimers and is unable to hydrolyze ATP while, when purified from the soluble fraction, TraB can form hexamers capable of hydrolyzing ATP. Remarkably, both the N- and C-terminal domains display ATP-hydrolyzing activity. These properties define a new class of VirB4 proteins.

Two-step and one-step secretion mechanisms in Gram-negative bacteria: contrasting the type IV secretion system and the chaperone-usher pathway of pilus biogenesis.

Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the one-step mechanism, whereby substrates are translocated directly from the bacterial cytoplasm to the extracellular medium or into the eukaryotic target cell; (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial inner membrane; once in the periplasm, substrates are targeted to one of the secretion systems that mediate transport across the outer membrane and released outside the bacterial cell. The present review provides an example for each of these two classes of secretion systems and contrasts the various solutions evolved to secrete substrates.

Protein modification, bioconjugation, and disulfide bridging using bromomaleimides.

The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein-maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.

The structural biology of type IV secretion systems.

Type IV secretion systems (T4SSs) are versatile secretion systems that are found in both Gram-negative and Gram-positive bacteria and secrete a wide range of substrates, from single proteins to protein-protein and protein-DNA complexes. They usually consist of 12 components that are organized into ATP-powered, double-membrane-spanning complexes. The structures of single soluble components or domains have been solved, but an understanding of how these structures come together has only recently begun to emerge. This Review focuses on the structural advances that have been made over the past 10 years and how the corresponding structural insights have helped to elucidate many of the details of the mechanism of type IV secretion.