Gabriel Waksman

Birkbeck, University of London, Londinium, England, United Kingdom

Are you Gabriel Waksman?

Claim your profile

Publications (129)1046.66 Total impact

  • Source
    Dataset: BMC Cover
  • Source
  • Source
  • Source
  • [Show abstract] [Hide abstract]
    ABSTRACT: PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue 'communities') within the translocation domain (especially around β12-β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability.
    eLife. 10/2014; 3.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel series of 8-amino imidazo[1,2-a]pyrazine derivatives has been developed as inhibitors of the VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imidazo[1,2-a]pyrazines has been used to probe the structure–activity relationships of these inhibitors, which show potential as antibacterial agents.
    Bioorganic & Medicinal Chemistry. 09/2014;
  • Source
    Tamir Gonen, Gabriel Waksman
    Current Opinion in Structural Biology 09/2014; · 8.74 Impact Factor
  • David Steadman, Alvin Lo, Gabriel Waksman, Han Remaut
    [Show abstract] [Hide abstract]
    ABSTRACT: The rise of multidrug resistant bacteria is a major worldwide health concern. There is currently an unmet need for the development of new and selective antibacterial drugs. Therapies that target and disarm the crucial virulence factors of pathogenic bacteria, while not actually killing the cells themselves, could prove to be vital for the treatment of numerous diseases. This article discusses the main surface architectures of pathogenic Gram-negative bacteria and the small molecules that have been discovered, which target their specific biogenesis pathways and/or actively block their virulence. The future perspective for the use of antivirulence compounds is also assessed.
    Future Microbiology 07/2014; 9:887-900. · 4.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Uropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) in approximately 50% of women. These bacteria use type 1 and P pili for host recognition and attachment. These pili are assembled by the chaperone-usher pathway of pilus biogenesis. The review examines the biogenesis and adhesion of the UPEC type 1 and P pili. Particular emphasis is drawn to the role of the outer membrane usher protein. The structural properties of the complete pilus are also examined to highlight the strength and functionality of the final assembly. The usher orchestrates the sequential addition of pilus subunits in a defined order. This process follows a subunit-incorporation cycle which consists of four steps: recruitment at the usher N-terminal domain, donor-strand exchange with the previously assembled subunit, transfer to the usher C-terminal domains and translocation of the nascent pilus. Adhesion by the type 1 and P pili is strengthened by the quaternary structure of their rod sections. The rod is endowed with spring-like properties which provide mechanical resistance against urine flow. The distal adhesins operate differently from one another, targeting receptors in a specific manner. The biogenesis and adhesion of type 1 and P pili are being therapeutically targeted, and efforts to prevent pilus growth or adherence are described. The combination of structural and biochemical study has led to the detailed mechanistic understanding of this membrane spanning nano-machine. This can now be exploited to design novel drugs able to inhibit virulence. This is vital in the present era of resurgent antibiotics resistance. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
    Biochimica et Biophysica Acta 05/2014; · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    Gabriel Waksman, Elena V Orlova
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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; · 9.81 Impact Factor
  • Sebastian Geibel, Gabriel Waksman
    [Show abstract] [Hide abstract]
    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
  • Source
    Trevor Lithgow, Gabriel Waksman
    [Show abstract] [Hide abstract]
    ABSTRACT: The EMBO conference 'From Structure to Function of Translocation Machines' took place in April 2013 in Dubrovnik, Croatia. The meeting brought together a mix of established and aspiring researchers to discuss a wealth of unpublished data and ideas in a lively scientific programme of talks designed to shatter the established dogma. From new ways to envisage known protein transport pathways to a brand new and totally unconventional protein transport system, surprises were part and parcel of this excellent EMBO conference.
    EMBO Reports 06/2013; · 7.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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; · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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

Publication Stats

4k Citations
1,046.66 Total Impact Points

Institutions

  • 2004–2014
    • Birkbeck, University of London
      • Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
    • The Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
    • Yale University
      • Department of Microbial Pathogenesis
      New Haven, CT, United States
  • 2013
    • Monash University (Australia)
      • Department of Biochemistry and Molecular Biology
      Melbourne, Victoria, Australia
  • 1997–2013
    • 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
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2011
    • Free University of Brussels
      • Structural Biology Brussels (SBB)
      Bruxelles, Brussels Capital Region, Belgium
    • 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