Asymmetric binding between SecA and SecB two symmetric proteins: implications for function in export.

SecB, a small tetrameric chaperone in Escherichia coli, facilitates export of precursor polypeptides from the cytoplasm to the periplasmic space. During this process, SecB displays two modes of binding. As a chaperone, it binds promiscuously to precursors to maintain them in a non-native conformation. SecB also demonstrates specific recognition of, and binding to, SecA. SecB with the precursor tightly bound enters an export-active complex with SecA and must pass the ligand to SecA at the translocon in the membrane. Here we use variants of SecA and SecB to further probe these interactions. We show that, unexpectedly, the binding between the two symmetric molecules is asymmetric and that the C-terminal alpha-helices of SecB bind in the interfacial region of the SecA dimer. We suggest that disruption of this interface by SecB facilitates conformational changes of SecA that are crucial to the transfer of the precursor from SecB to SecA.

Sites of interaction between SecA and the chaperone SecB, two proteins involved in export.

SecB, a small tetrameric cytosolic chaperone in Escherichia coli, facilitates the export of precursor poly-peptides by maintaining them in a nonnative conformation and passing them to SecA, which is a peripheral member of the membrane-bound translocation apparatus. It has been proposed by several laboratories that as SecA interacts with various components along the export pathway, it undergoes conformational changes that are crucial to its function. Here we report details of molecular interactions between SecA and SecB, which may serve as conformational switches. One site of interaction involves the final C-terminal 21 amino acids of SecA, which are positively charged and contain zinc. The C terminus of each subunit of the SecA dimer makes contact with the flat beta-sheet that is formed by each dimer of the SecB tetramer. Here we demonstrate that a second interaction exists between the extreme C-terminal alpha-helix of SecB and a site on SecA, as yet undefined but different from the C terminus of SecA. We investigated the energetics of the interactions by titration calorimetry and characterized the hydrodynamic properties of complexes stabilized by both interactions or each interaction singly using sedimentation velocity centrifugation.