Allosteric Regulation of SecA

Gold, Vicki A. M.; Robson, Alice; Clarke, Anthony R.; Collinson, Ian

doi:10.1074/jbc.m702066200

Cited by 44 publications

(26 citation statements)

References 45 publications

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“…Some studies using deletion constructs suggested that the N-terminus is part of the SecA dimerization interface, 20,21,25,76,77 whereas others concluded the opposite. 78,79 To address this issue, we deleted residues 2–8, 2–9, and 2–10 (Δ2–8, Δ2–9, and Δ2–10, respectively) from wild-type SecA and examined the effects on dimerization. SecA dimerization affinity decreases with the number of residues deleted from the N-terminus (Table 3).…”

Section: Resultsmentioning

confidence: 99%

Analysis of SecA Dimerization in Solution

et al. 2014

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The Sec pathway mediates translocation of protein across the inner membrane of bacteria. SecA is a motor protein that drives translocation of preprotein through the SecYEG channel. SecA reversibly dimerizes under physiological conditions, but different dimer interfaces have been observed in SecA crystal structures. Here, we have used biophysical approaches to address the nature of the SecA dimer that exists in solution. We have taken advantage of the extreme salt sensitivity of SecA dimerization to compare the rates of hydrogen–deuterium exchange of the monomer and dimer and have analyzed the effects of single-alanine substitutions on dimerization affinity. Our results support the antiparallel dimer arrangement observed in one of the crystal structures of Bacillus subtilis SecA. Additional residues lying within the preprotein binding domain and the C-terminus are also protected from exchange upon dimerization, indicating linkage to a conformational transition of the preprotein binding domain from an open to a closed state. In agreement with this interpretation, normal mode analysis demonstrates that the SecA dimer interface influences the global dynamics of SecA such that dimerization stabilizes the closed conformation.

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Section: Resultsmentioning

confidence: 99%

Analysis of SecA Dimerization in Solution

et al. 2014

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“…Cloning, expression and purification of the translocation components and specific mutants thereof were conducted as described previously [9,23]. Reconstitution of SecYEG into total Escherichia coli polar lipids has been well documented [9].…”

Section: Methodsmentioning

confidence: 99%

The dynamic action of SecA during the initiation of protein translocation

Gold

Whitehouse

Robson

et al. 2013

Biochemical Journal

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The motor ATPase SecA drives protein secretion through the bacterial Sec complex. The PPXD (pre-protein cross-linking domain) of the enzyme has been observed in different positions, effectively opening and closing a clamp for the polypeptide substrate. We set out to explore the implicated dynamic role of the PPXD in protein translocation by examining the effects of its immobilization, either in the position occupied in SecA alone with the clamp held open or when in complex with SecYEG with the clamp closed. We show that the conformational change from the former to the latter is necessary for high-affinity association with SecYEG and a corresponding activation of ATPase activity, presumably due to the PPXD contacting the NBDs (nucleotide-binding domains). In either state, the immobilization prevents pre-protein transport. However, when the PPXD was attached to an alternative position in the associated SecYEG complex, with the clamp closed, the transport capability was preserved. Therefore large-scale conformational changes of this domain are required for the initiation process, but not for translocation itself. The results allow us to refine a model for protein translocation, in which the mobility of the PPXD facilitates the transfer of pre-protein from SecA to SecYEG.

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“…Steady-state SecA ATPase measurements were monitored at 25 °C using a pyruvate kinase (PK)/lactate dehydrogenase (LDH)-coupled assay as previously described (18). Data were fitted to a one-site quadratic tight ligand-binding equation with background, Equation 1 defined as, where v is equal to enzyme velocity, B max is the total capacity of SecA-ligand, [ L ] is the total ligand ( i.e.…”

Section: Methodsmentioning

confidence: 99%

“…Crystal structures have been obtained both for dimers of SecA (15) and for monomers of the enzyme (16), but dimers predominate in solution (17, 18). The structure of SecA bound to SecYEG in a 1:1 stoichiometry in the presence of an ATP analog (ADP-BeF x or ADP-AlF x ), reveals a major conformational change in the PPXD, which rotates away from the HWD to contact NBD2, in comparison with the structure of SecA alone (4).…”

Section: Introductionmentioning

confidence: 99%

The Oligomeric State and Arrangement of the Active Bacterial Translocon

Deville

Gold

Robson

et al. 2011

Journal of Biological Chemistry

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116

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Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that while monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.

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Allosteric Regulation of SecA

Cited by 44 publications

References 45 publications

Analysis of SecA Dimerization in Solution

Analysis of SecA Dimerization in Solution

The dynamic action of SecA during the initiation of protein translocation

The Oligomeric State and Arrangement of the Active Bacterial Translocon

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