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Donald Oliver

Publications and source records attributed to Donald Oliver.

7 recordsLinked to original sources
PubMed2006

SecA dimer cross-linked at its subunit interface is functional for protein translocation.

SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.

Adenosine Triphosphatases↗
PubMed2005

Dimeric SecA is essential for protein translocation.

SecA facilitates bacterial protein translocation by its association with presecretory or membrane proteins and the SecYEG translocon channel. Once assembled, SecA ATPase undergoes cycles of membrane insertion and retraction at SecYEG that drive protein translocation in a stepwise fashion. SecA exists in equilibrium between a monomer and dimer, and association with its translocation ligands shifts this equilibrium dramatically. Here, we examined the proposal that protein translocation can occur by means of a SecA monomer. We produced a mutant SecA protein lacking residues 2-11, which was found to exist mostly as a monomer, and it was unable to complement a conditional-lethal secA mutant, was inactive for in vitro protein translocation, and was poorly active for translocation ATPase activity. Furthermore, we developed a technique termed membrane trapping, where wild-type SecA subunits became trapped within the membrane by overproduction of membrane-stuck mutant SecA proteins, and, in one case, a membrane-associated SecA heterodimer was demonstrated. Finally, we examined both endogenous and reconstituted membrane-bound SecA and found a significant level of SecA dimer in both cases, as assessed by chemical crosslinking. Collectively, our results strongly suggest that membrane-bound SecA dimer is critical for the protein translocation cycle, although these results cannot exclude participation of SecA monomer at some stage in the translocation process. Our findings have important implications regarding SecA motor function and translocon assembly and activation.

Adenosine Triphosphatases↗
PubMed2003

Nucleotide and phospholipid-dependent control of PPXD and C-domain association for SecA ATPase.

The SecA ATPase motor is a central component of the eubacterial protein translocation machinery. It is comprised of N- and C-domain substructures, where the N-domain is comprised of two nucleotide-binding domains that flank a preprotein-binding domain (PPXD), while the C-domain binds phospholipids as well as SecB chaperone. Our recent crystal structure of Bacillus subtilis SecA protomer [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] along with experimental support for the correct dimer structure [Ding, H., Hunt, J. F., Mukerji, I., and Oliver, D. (2003) Biochemistry 42, 8729-8738] have now allowed us to study SecA structural dynamics during interaction with various translocation ligands and to relate these findings to current models of SecA-dependent protein translocation. In this paper, we utilized fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues within the PPXD and C-domains of SecA to investigate the interaction of these two domains and their response to temperature, model membranes, and nucleotide. Consistent with the crystal structure of SecA, we found that the PPXD and C-domains are proximal to one another in the ground state. Increasing temperature or binding to model membranes promoted a loosening of PPXD and C-domain association, while ADP binding promoted a tighter association. A similar pattern of PPXD and C-domain association was obtained also for Escherichia coli SecA protein. Furthermore, a hyperactive Azi-PrlD SecA protein of E. coli had increased PPXD and C-domain separation, consistent with its activation in the ground state. Interestingly, PPXD and C-domain separation occurred prior to the onset of major temperature-induced conformational changes in both the PPXD and C-domains of SecA. Our results support a model in which PPXD and C-domain proximity is important for regulating the initial stages of SecA activation, and they serve also as a template for future structural studies aimed at elucidation of the chemomechanical cycle of SecA-dependent protein translocation.

Adenosine Diphosphate↗
PubMed2003

Two-stage binding of SecA to the bacterial translocon regulates ribosome-translocon interaction.

The bacterial translocon interacts with both SecA-bound preproteins and nascent chain-ribosome complexes during Sec and signal recognition particle-dependent protein translocation, respectively. In their inactive state, translocons are saturated with ribosomes and SecA protein, reflecting the inherent affinity of these components for one another. We found that SecA and ribosomes are bound simultaneously and noncompetitively to a common set of inactive translocons. Furthermore, we demonstrate that at a later stage in binding, SecA possesses a ribosome-translocon dissociation activity that is coupled to its ATP-dependent membrane insertion and retraction cycle that drives protein translocation. This novel activity is presumably important in the commitment of the translocon to the Sec-dependent pathway. These results also provide a rationale for the compatibility and regulation of multiple protein translocation pathways that each makes distinct demands on a common translocon core.

Adenosine Triphosphatases↗
PubMed2003

Bacillus subtilis SecA ATPase exists as an antiparallel dimer in solution.

SecA ATPase promotes the biogenesis of membrane and secretory proteins into and across the cytoplasmic membrane of Eubacteria. SecA binds to translocon component SecYE and substrate proteins and undergoes ATP-dependent conformational cycles that are coupled to the stepwise translocation of proteins. Our recent crystal structure of B. subtilis SecA [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] showed two different dimer interactions in the lattice which both buried significant solvent-accessible surface area in their interface and could potentially be responsible for formation of the physiological dimer in solution. In this paper, we utilize fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues to determine the oligomeric structure of SecA protein in solution. Our results show that of the two dimers interactions observed in the crystal structure, SecA forms an antiparallel dimer in solution that maximizes the buried solvent-accessible surface area and intermolecular contacts. At the submicromolar protein concentrations used in the fluorescence experiments, we saw no evidence for the formation of higher-order oligomers of SecA based on either the alternative dimer or the 3(1) helical fiber observed in the crystal lattice. Our studies are consistent with previous ones demonstrating the existence of a dimerization determinant within the C-domain of SecA as well as those documenting the interaction of N- and C-domains of SecA. Our results also provide a valuable starting point for a determination of whether the subunit status of SecA changes during the protein translocation as well as studies designed to elucidate the conformational dynamics of this multidomain protein during its translocation cycle.

Adenosine Triphosphatases↗
PubMed2002

Critical regions of secM that control its translation and secretion and promote secretion-specific secA regulation.

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.

3' Untranslated Regions↗