Abstract
SecA, the dimeric ATPase subunit of protein translocase, contains a DEAD helicase catalytic core that binds to a regulatory C-terminal domain. We now demonstrate that IRA1, a conserved helix-loop-helix structure in the C-domain, controls C-domain conformation through direct interdomain contacts. C-domain conformational changes are transmitted to the DEAD motor and alter its conformation. These interactions establish DEAD motor/C-domain conformational cross-talk that requires a functional IRA1. IRA1-controlled binding/release cycles of the C-domain to the DEAD motor couple this cross-talk to protein translocation chemistries, i.e. DEAD motor affinities for ligands (nucleotides, preprotein signal peptides, and SecYEG, the integral membrane component of translocase) and ATP turnover. IRA1-mediated global co-ordination of SecA catalysis is essential for protein translocation.
Highlights
C-domain, controls C-domain conformation through direct interdomain contacts
Long bent ␣-helix, which docks the C-domain to the DEAD motor by acting as a molecular staple binding both nucleotide binding domain (NBD) and intramolecular regulator of ATPase2 (IRA2); the flexible wing domain (WD); IRA1 (15), a conserved helix-loophelix (H1-L-H2) that fits between scaffold domain (SD) and substrate specificity domain (SSD) (Fig. 5); and the extreme C-terminal region (CTD), which is largely crystallographically unresolved and binds lipid and SecB (26, 27)
We propose that SecA ATP binding and hydrolysis become coupled to protein translocation through IRA1 acting as a global co-ordinator of translocase catalysis and conformation
Summary
C-domain, controls C-domain conformation through direct interdomain contacts. C-domain conformational changes are transmitted to the DEAD motor and alter its conformation. The DEAD motor contains two “RecA-like” subunregulated, hyperactivated ATPase that is incompetent for translocation (15) This observation led us to propose that IRA1 is a molecular switch essential for coupling ATP hydrolysis to translocation work (15). We show that IRA1 contacts other SecA subdomains and through these it controls association and conformational cross-talk between the DEAD motor and the C-domain. Modulation of these physical contacts allows IRA1 to regulate DEAD motor subactivities. We propose that SecA ATP binding and hydrolysis become coupled to protein translocation through IRA1 acting as a global co-ordinator of translocase catalysis and conformation
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