Analysis of SecA Dimerization in Solution

Abstract

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.

Stabilization of SecA ATPase by the primary cytoplasmic salt of Escherichia coli

“…, black curves). In 500 m M KCl at 22°C, SecA exists mostly (90%) as monomers, as expected from the analytical ultracentrifuge measurements of Wowor et al But at 37°, the equilibrium shifts toward dimers, suggesting that the dimerization interface involves hydrophobic as well as electrostatic interactions. Results from measurements in KGlu (Fig.…”

“…Analytical ultracentrifuge studies of SecA show that dimerization depends strongly on ionic strength . For example, in 100 m M KCl about 90% of SecA is dimeric while in 500 m M KCl, about 95% is monomeric . Although there are wide variations in buffer compositions reported in the literature, SecA is typically studied as a dimer in buffered chloride solutions.…”

“…10 For example, in 100 mM KCl about 90% of SecA is dimeric while in 500 mM KCl, about 95% is monomeric. 12 Although there are wide variations in buffer compositions reported in the literature, SecA is typically studied as a dimer in buffered chloride solutions. Using 50 mM KCl and SecA extracted from E. coli, Song and Kim 13 observed through tryptophan fluorescence measurements that SecA tertiary structure was 50% destabilized at 37 C while CD spectroscopy revealed a complete unfolding transition with a midpoint of about 45 C. Similar unfolding temperatures had been observed earlier by Ulbrandt et al 14 Den Blaauwen et al 15 found similar transition temperatures by calorimetry for B. subtilis SecA.…”

Novel Sequence Feature of SecA Translocase Protein Unique to the Thermophilic Bacteria: Bioinformatics Analyses to Investigate Their Potential Roles

“…Earlier structural and functional studies also indicate that SecA can adopt a wide variety of oligomeric states [102][103][104][105] and it is possible that the presence of these CSI could stabilize certain oligomeric forms that are of relevance for the functioning of this protein in a thermophilic environment. In addition, most thermophilic and hyperthermophilic bacteria lacks SecB, a molecular chaperone protein which plays an important role in transferring pre-protein to SecA and SecYEG translocation system in most other bacteria [106][107][108].…”

Applications of Hydrogen/Deuterium Exchange MS from 2012 to 2014

“… 52 Further HDX MS analysis of SecA focused on dimerization and how mutation would disrupt formation of the dimer. 53 Mutagenesis in exchange of a protein activated by cAMP (EPAC) showed that a single mutation can shift the conformational dynamics toward the extended active conformation. 54 …”