The characterization of SecA and its interactions with signal sequences in the bacterial secretory pathway

Signal sequence recognition by SecA, a preprotein translocating molecular machine, is a major and unique cellular function in the bacterial secretory pathway. The SecA ATPase must specifically and efficiently recognize the amino-terminal signal sequences of newly synthesized polypeptides to be able to target them to the cytoplasmic membrane. To elucidate the basis of SecA mediated signal sequence recognition and the details of SecA driven preprotein translocation, biophysical and biochemical characterizations of structure-function relationship and the interactions between SecA and signal sequences have been performed and addressed in this dissertation. Our NMR study of Escherichia coli SecA demonstrates that SecA has two major assigned mobile regions: one is located within the second nucleotide-binding fold (NBF-II; residues 564–579) and the other is the extreme C-terminal segment of SecA (residues 864–901). Both mobile regions are essential for preprotein translocation activity and involved in functionally critical regulatory steps in the mechanism of SecA. Chemical cross-linking studies show that the oligomeric state of SecA in solution is altered by the presence of signal sequences and phospholipids. Addition of functional signal peptides shifts the equilibrium of the oligomeric state of SecA from the major dimeric state to the higher oligomers. In contrast, addition of phospholipids shifts the equilibrium to favor the more monomers. These results suggest that the oligomeric state of SecA may change dynamically during the translocation reaction and imply that bacteria may utilize the cycles of SecA subunit recruitment and dissociation to achieve efficient processive translocation. Lastly, the NMR study of a SecA-bound signal peptide reveals that this peptide forms an α-helical conformation in its hydrophobic h-region and extended conformations in the n- and c-regions when bound to SecA. Line-broadening effects indicate that the positively charged arginine residues of the n-region and the hydrophobic residues of the h-region appear to be more immobilized than the polar residues of the c-region during binding to SecA. TrNOEs suggest that the V15/A18/V21 side of the helical h-region is more strongly bound in the binding pocket. Therefore, the positively charged n-region and the hydrophobic helical h-region could be the selective features needed for signal sequence recognition by SecA