The Seca Dimer Interface

Abstract

As a central component in the general secretion (Sec) pathway of bacteria, the ATPase motor protein, SecA, mediates preprotein translocation through the integral membrane channel, SecYEG. SecA is a potential target for antibacterial therapeutics because it is crucial for protein transport and cell viability. It is highly conserved among species of bacteria, yet it has no close human homologs. In the Sec pathway, SecA interacts with various ligands, including other SecA molecules. The latter interaction, a monomer-dimer equilibrium, is highly sensitive to salt concentration, temperature, and ligand binding. Although the structure of the SecA protomer is well-conserved among bacterial homologs, multiple dimer interfaces have been identified in SecA dimer crystal structures. We employed several biophysical methods to define the SecA dimer interface in solution and map the dimer interface using strategies independent of those used to solve the crystal structures of SecA dimer. By measuring the effects of alanine substitution on dimerization energetics using sedimentation velocity analytical ultracentrifugation, we determined that the substitution of residues at the N-terminus and within the helical scaffold domain significantly decreases dimerization affinity. By monitoring the backbone hydrogen/deuterium exchange rates using mass spectrometry, we found that residues lying within the helical scaffold domain are protected from exchange in the dimer state, consistent with the analytical ultracentrifugation results. These data are consistent with the Bacillus subtilis (1M6N) and Thermus thermophilus (2IPC) SecA dimeric structures.