CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, the Type IV competence pilus is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to ~2.7 Å. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target

CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, the Type IV competence pilus is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to ~2.7 Å. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target

CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation

Characterization and Structure of a Zn2+ and [2Fe-2S]-containing Copper Chaperone from Archaeoglobus Fulgidus

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine rich N-terminal domain of 130 amino acids in addition to a C-terminal copper-binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and X-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 Å resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking

Structural studies of BMMs

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2004.Vita.Includes bibliographical references.(cont.) α-subunit cavities. The presence of 6-bromohexan-l-ol induces one of the active site helices to adopt a [pi] conformation. Together, these findings suggest modes by which molecules may move through the MMOH cavities and how both substrates and MMOB may influence the structure of the active site pocket.Bacterial multicomponent monooxygenases (BMMs) are capable of oxidizing a variety of hydrocarbon substrates at a non-heme carboxylate-bridged diiron center housed within a 200-250 kDa hydroxyase protein. Chapter 1 introduces the members of the BMM family as well as several related diiron proteins with functional relevance to BMMs. The structures of the individual components and the diiron centers are discussed in relation to their catalytic function and the tuning of the metal centers. The structure of the toluene /o-xylene monooxygenase hydroxylase (ToMOH) is presented in chapter 2. The dinuclear iron center is virtually identical to that in the methane monooxygenase hydroxylase (MMOH), yet several novel features, such as a 40 [angstrom] channel, may explain the differences in the substrate specificity between BMM subfamily members. A structural basis for the regiospecificities of toluene monooxygenase and phenol hydroxylases is discussed In Chapter 3 are described metal reconstitution studies of MMOH to probe the ligand geometries of the diiron center and the possible effects on the structure by the coupling protein, MMOB, and the orfY gene product, MMOD. The structures of Mn(II) and Co(II) reconstituted MMOH are identical to that of the diferrous protein. MMOB and MMOD make the addition and removal of iron from MMOH more difficult, suggesting that these proteins serve to block solvent and/or small molecule access to the active site by binding to the four-helix bundle housing the diiron center. Product movement to and from the diiron centers of BMMs is essential for catalytic function. In chapter 4 the crystal structures of MMOH with several bound products are reported. The binding of these products alter the positioning of several side chains in the MMOHby Matthew H. Sazinsky.Ph.D

CryoEM Structure of the Vibrio cholerae Type IV Pilus Secretin PilQ

Natural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV pilus (T4P) is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound T4P retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.5 Å and provide insight on the channel DNA may traverse through during uptake

CryoEM Structure of the Vibrio cholerae Type IV Pilus Secretin PilQ

Natural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV pilus (T4P) is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound T4P retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.5 Å and provide insight on the channel DNA may traverse through during uptake

Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD-Transfer Reaction

The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FAD_ox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s^–1, in a step that is coupled to the rapid dissociation of NAD⁺. Electrochemical and equilibrium-binding studies indicate that NSMOA binds FAD_hq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FAD_hq from the reductase to the epoxidase. After binding to NSMOA, FAD_hq rapidly reacts with molecular oxygen to form a stable C­(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein–protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD⁺ after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FAD_hq to NSMOA. The binding of pyridine nucleotides to SMOB–FAD_hq shifts the FAD_hq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB–FAD_ox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions