Linkage Between SecA Dimerization and Ligand Binding

Abstract

The general secretion (Sec) pathway, found in bacteria, archaea, and eukaryotes, transports preproteins across membranes. The SecA protein mediates preprotein translocation through the SecYEG channel linked to ATP hydrolysis. Several studies suggest that SecA exists in a monomer-dimer equilibrium. Although self-association of SecA has been intensively studied, the oligomeric state of SecA, especially during preprotein transport, remains controversial. SecA dimerization is reported to be sensitive to salt concentration, temperature and ligand binding. We have characterized the energetics of SecA dimerization as a function of salt concentration, temperature and binding of a signal peptide using sedimentation velocity analytical ultracentrifugation. We employ fluorescence-detection to enhance sensitivity at low protein concentrations. SecA was labeled with Alexa Fluor 488 at the N-terminus. Labeling does not affect the dimer dissociation constant. Lower salt concentrations and higher temperatures greatly enhance dimerization. The dimer dissociation constants measured at 20°C range from 40 µM in 500 mM KCl to 14 nM in 100 mM KCl. Linkage analysis indicates that SecA dimerization is accompanied by the release of 5 ions. In addition, SecA dimerization is reduced upon binding of signal peptide, indicating that SecA oligomerization and ligand binding are thermodynamically linked.