Gabriel Waksman

The Institute of Structural and Molecular Biology, Londinium, England, United Kingdom

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Publications (166)1396.67 Total impact

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    ABSTRACT: 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 1MegaDalton (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.
    Current Opinion in Structural Biology 04/2014; 27C:16-23. · 8.74 Impact Factor
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    ABSTRACT: 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.
    Nature 03/2014; · 38.60 Impact Factor
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    Gabriel Waksman, Elena V Orlova
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    ABSTRACT: Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNAs and/or proteins through the membranes of bacteria. Because of their complexity and multi-protein organisation, T4S systems have been extremely challenging to study structurally. However in the past five years significant milestones have been achieved by X-ray crystallography and cryo-electron microscopy. This review describes some of the more recent advances: the structures of some of the protein components of the T4S systems and the complete core complex structure that was determined using electron microscopy.
    Current opinion in microbiology 02/2014; 17C:24-31. · 7.87 Impact Factor
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    ABSTRACT: 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 ∼3 MDa 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.
    Current Opinion in Structural Biology 01/2014; 27:16–23. · 8.74 Impact Factor
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    ABSTRACT: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). 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. 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. 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.
    Journal of Antimicrobial Chemotherapy 12/2013; · 5.34 Impact Factor
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    ABSTRACT: 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 β-barrel translocation domain (TD), a β-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 (β12-13 loop) was found to facilitate pore opening. Mutation of the β12-13 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.
    Proceedings of the National Academy of Sciences 12/2013; · 9.74 Impact Factor
  • Sebastian Geibel, Gabriel Waksman
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    ABSTRACT: Secretion systems are specialized in transport of proteins, DNA or nutrients across the cell envelope of bacteria and enable them to communicate with their environment. The chaperone-usher (CU) pathway is used for assembly and secretion of a large family of long adhesive protein polymers, termed pili, and is widespread among Gram-negative pathogens [1]. Moreover, recent evidence has indicated that CU secretion systems are also involved in sporulation [2,3]. In this review we focus on the structural biology of the paradigmatic type 1 and P pili CU systems encoded by uropathogenic Escherichia coli (UPEC), where recent progress has provided unprecedented insights into pilus assembly and secretion mechanism. This article is part of a Special Issue entitled:Protein trafficking & Secretion.
    Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
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    ABSTRACT: Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three sub-domains with striking structural homology with helicase sub-domains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities.
    Molecular Microbiology 05/2013; · 4.96 Impact Factor
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    ABSTRACT: Type 1 pili, produced by uropathogenic Escherichia coli, are multisubunit fibres crucial in recognition of and adhesion to host tissues. During pilus biogenesis, subunits are recruited to an outer membrane assembly platform, the FimD usher, which catalyses their polymerization and mediates pilus secretion. The recent determination of the crystal structure of an initiation complex provided insight into the initiation step of pilus biogenesis resulting in pore activation, but very little is known about the elongation steps that follow. Here, to address this question, we determine the structure of an elongation complex in which the tip complex assembly composed of FimC, FimF, FimG and FimH passes through FimD. This structure demonstrates the conformational changes required to prevent backsliding of the nascent pilus through the FimD pore and also reveals unexpected properties of the usher pore. We show that the circular binding interface between the pore lumen and the folded substrate participates in transport by defining a low-energy pathway along which the nascent pilus polymer is guided during secretion.
    Nature 04/2013; 496(7444):243-6. · 38.60 Impact Factor
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    ABSTRACT: DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single molecule and X-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high-precision FRET we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed ″nucleotide binding″ state predominates. Photon distribution and hidden Markov analysis revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the ″nucleotide binding″ state: After nucleotide binding, correct dNTPs are transported to the closed state while incorrect dNTPs are delivered to the open state.
    Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
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    ABSTRACT: Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.
    The EMBO Journal 03/2013; · 9.82 Impact Factor
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    ABSTRACT: Type 1 pili are representative of a class of bacterial surface structures assembled by the conserved chaperone/usher pathway and used by uropathogenic Escherichia coli to attach to bladder cells during infection. The outer membrane assembly platform - the usher - is critical for the formation of pili, catalysing the polymerization of pilus subunits and enabling the secretion of the nascent pilus. Despite extensive structural characterisation of the usher, a number of questions about its mechanism remain, notably its oligomerization state, and how it orchestrates the ordered assembly of pilus subunits. We demonstrate here that the FimD usher is able to catalyse in vitro pilus assembly effectively in its monomeric form. Further, by establishing the kinetics of usher-catalysed reactions between various pilus subunits, we establish a complete kinetic model of tip fibrillum assembly, able to account for the order of subunits in native type 1 pili.
    Journal of Molecular Biology 01/2013; · 3.91 Impact Factor
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    ABSTRACT: Klebsiella species are members of the family enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. Among other virulence factors in Klebsiella, type 3 pili exhibit a unique binding pattern in the human kidney via interaction of two MrkD adhesion variants 1C1 and 1P to type IV and/or V collagen. However, very little is known about the nature of this recognition. Here we present the crystal structure of the plasmid born MrkD(1P) receptor domain (MrkDrd). The structure reveals a jelly-roll β-barrel fold comprising 17 β-strands very similar to the receptor domain of GafD, the tip adhesin from the F17 pilus that recognizes n-acetyl-d-glucosamine (GlcNAc). Analysis of collagen V binding of different MrkD(1P) mutants revealed that two regions were responsible for its binding: a pocket, that aligns approximately with the GlcNAc binding pocket of GafD involving residues R105 and Y155, and a transversally oriented patch that spans strands β2a, β9b and β6 including residues V49, T52, V91, R102 and I136. Taken together, these data provide structural and functional insights on MrkD(1P) recognition of host cells, providing a tool for future development of rationally designed drugs with the prospect of blocking Klebsiella adhesion to collagen V.
    Molecular Microbiology 09/2012; · 4.96 Impact Factor
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    Tamir Gonen, Gabriel Waksman
    Current Opinion in Structural Biology 07/2012; 22(4):467-8. · 8.74 Impact Factor
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    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 06/2012; 109(28):11348-53. · 9.74 Impact Factor
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    ABSTRACT: P pili are prototypical chaperone-usher pathway-assembled pili used by Gram-negative bacteria to adhere to host tissues. The PapC usher contains five functional domains: a transmembrane β-barrel, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we delineated usher domain interactions between themselves and with chaperone-subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone-adhesin-bound usher has its Plug displaced from the pore, adjacent to the NTD. We demonstrate an interaction between the NTD and Plug domains that suggests a biophysical basis for usher gating. Furthermore, we found that the NTD exhibits high-affinity binding to the chaperone-adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). We also demonstrate that CTD2 binds with lower affinity to all tested chaperone-subunit complexes except for the chaperone-terminator subunit (PapDH) and has a catalytic role in dissociating the NTD-PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTD-Plug complex bound all of the chaperone-subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone-subunit complexes to the usher and is the sole recruiter of PapDH. Overall, our studies reveal the cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone-usher pathway pilus.
    Proceedings of the National Academy of Sciences 05/2012; 109(24):9563-8. · 9.74 Impact Factor
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    Journal of Molecular Biology. 05/2012; 418(s 3–4):264–265.
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    Gabriel Waksman
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    ABSTRACT: Bacterial secretion systems play essential roles in pathogenesis and also in maintaining lines of communication between bacterial cells in the bacterial microflora or between commensal bacteria and their host. Recent breakthroughs in the field have yielded some novel insights into the mechanisms by which these systems operate. This issue of Philosophical Transactions B seeks to provide a detailed survey of the field, with an emphasis on mechanisms and how their unravelling might provide a new future for antibiotics research.
    Philosophical Transactions of The Royal Society B Biological Sciences 04/2012; 367(1592):1014-5. · 6.23 Impact Factor
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    Andreas Busch, Gabriel Waksman
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    ABSTRACT: Up to eight different types of secretion systems, and several more subtypes, have been described in Gram-negative bacteria. Here, we focus on the diversity and assembly mechanism of one of the best-studied secretion systems, the widespread chaperone-usher pathway known to assemble and secrete adhesive surface structures, called pili or fimbriae, which play essential roles in targeting bacterial pathogens to the host.
    Philosophical Transactions of The Royal Society B Biological Sciences 04/2012; 367(1592):1112-22. · 6.23 Impact Factor
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    William J Allen, Gilles Phan, Gabriel Waksman
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    ABSTRACT: Pili belong to a broad class of bacterial surface structures that play a key role in infection and pathogenicity. The largest and best characterised pilus biogenesis system--the chaperone-usher pathway--is particularly remarkable in its ability to synthesise and display highly organised structures at the outer membrane without any input from endogenous energy sources. The past few years have heralded exciting new developments in our understanding of the structural biology and mechanism of pilus assembly, which are discussed in this review. Such knowledge will be particularly important in the future, as we approach an era of widespread resistance to common antibiotics and require new targets.
    Current Opinion in Structural Biology 03/2012; 22(4):500-6. · 8.74 Impact Factor

Publication Stats

7k Citations
1,396.67 Total Impact Points

Institutions

  • 2004–2014
    • The Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
    • Birkbeck, University of London
      • Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
    • Yale University
      • Department of Microbial Pathogenesis
      New Haven, CT, United States
  • 1994–2013
    • Washington University in St. Louis
      • • Department of Biochemistry and Molecular Biophysics
      • • Department of Pathology and Immunology
      • • Department of Pediatrics
      San Luis, Missouri, United States
  • 2012
    • University of Washington Seattle
      • Department of Immunology
      Seattle, WA, United States
  • 1999–2012
    • Howard Hughes Medical Institute
      Maryland, United States
  • 2011
    • University of Lyon
      Lyons, Rhône-Alpes, France
  • 2010
    • MRC National Institute for Medical Research
      • Division of Molecular Structure
      Londinium, England, United Kingdom
  • 2009
    • Duke University Medical Center
      • Department of Pediatrics
      Durham, NC, United States
  • 2008
    • Otto-von-Guericke-Universität Magdeburg
      • Institute for Medical Microbiology
      Magdeburg, Saxony-Anhalt, Germany
    • University Pompeu Fabra
      Barcino, Catalonia, Spain
  • 2004–2008
    • University College London
      • Department of Structural and Molecular Biology
      London, ENG, United Kingdom