Elucidating the Dimer Interface of Seca

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SecA is an ATPase that mediates preprotein translocation through the SecYEG channel. 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, and it has no close human homologs. As a central component in the general secretion pathway of bacteria, SecA interacts with various ligands, including other SecA molecules. SecA exists in a monomer-dimer equilibrium at micromolar concentrations that is highly sensitive to salt concentration and temperature. Although the structure of the SecA protomer is well-conserved among bacterial homologs, multiple dimer interfaces have been identified. To define the physiological dimer interface of SecA, we have performed site-directed mutagenesis based on the alternative dimer interfaces reported in the crystal structures. Residues for mutagenesis were chosen by computational alanine scanning using the program Robetta. The selected mutations were predicted to destabilize the interface by at least 1 kcal/mol. By using sedimentation velocity, we determined the effect of alanine substitution on dimerization energetics. We have identified four residues that substantially affect dimerization.