Allosteric Communication between Signal Peptides and the SecA Protein DEAD Motor ATPase Domain

Abstract

SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain. SecA recognizes mature and signal peptide preprotein regions. We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding. Binding is abrogated by deletion of residues 219 -244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain. SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins. Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch. IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity. This mechanism coordinates signal peptide binding with ATPase activation. Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase. This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants. Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.Several cellular polypeptides cross biological membranes prior to acquiring their native state. Such proteins are delivered by chaperone-like factors (e.g. signal recognition particle and SecB) (1, 2) to membranes and subsequently cross them through specialized pumps termed preprotein translocases or translocons (3-5). The bacterial Sec translocase comprises the membrane proteins SecYEGDFYajC and the peripheral ATPase SecA (4, 5). Several of these components are essential and conserved in the three domains of life. Secretory proteins bind to the SecA motor and activate its ATPase. This triggers SecA "insertion-deinsertion" cycles at SecYEG (6, 7), allowing processive translocase movement along the polymeric substrate (8) in defined steps (9, 10). Substrates are thought to transverse the bilayer through a putative "pore" formed by the essential SecYEA core (11-13). Proton motive force (14), SecG (15, 16), and SecDF (8, 16) regulate SecA cycling.Dimeric SecA is built of defined mechanical parts (17, 18). Each protomer (102 kDa) comprises a 68-kDa N-terminal domain (N68) homologous to ATPase motor domains of DEAD helicases (8,18,19) and a C-terminal (C34) dimerization domain (17,20). The SecA DEAD motor forms a proposed mononucleotide binding fold (18, 21) built from an amino-terminal region harboring a nucleotide binding subdomain (NBD) that contains Walker box A and B sequences (DEAD helicase motifs I and II; Fig. 2A) (22) and the intramolecular regulator of ATP hydrolysis (IRA2) subdomain (18). N68 displays a high, unregulated ATPase activity (17).Deletion and point mutants helped determine the basic features of SecA catalysis (17, 18). IRA1, an essentiaI molecular switch in C34, regulates the DEAD motor ATPase through C34/DEAD ...

Use of synthetic signal sequences to explore the protein export machinery

“…Using fragments, deletional analysis, and chemical crosslinking of synthetic signal peptides, Economou's group concluded that the region at the base of the PPXD (so-called stem region) serves to bind the signal sequence. 69,89 Results of a FRET-based study with complementary crosslinking approaches also implicated the PPXD (specifically the third Using Signal Peptides to Explore Protein Export 315 helix) in signal sequence binding, 93,95 which is consistent with the early crosslinking studies from Mizushima's lab. 94 We have performed a sequence alignment of 550 SecA proteins, and the region proposed by Musial-Siwek et al 93 has a low conservation score (J. L. Maki and L. M. Gierasch, unpublished observations), raising doubt about whether it serves as a direct binding site for signal sequences.…”

“…They also identified another region that had synergistic effects, and termed it IRA2 and the first segment IRA1. 69,70 IRA2, in fact, is the second helicase domain, which was clear once the crystal structure was available.…”

“…Studies from a variety of groups over the past decade have revealed that SecA is a multidomain protein that can exist as a monomer or a dimer, and that its domain rearrangements can be triggered by binding to any of its several ligands. 67,[69][70][71][72][73][74][75][76][77][78][79][80][81] The key to the conformational rearrangements appears to be intramolecular interactions that stabilize a more tightly packed, ''closed'' form of the protein. Weakening these interactions relieves these intramolecular constraints, and the protein domains partially dissociate, creating a less tightly packed, ''open'' form of the protein.…”

“…75,76,79,80 Thus, the picture that emerges is that the ligand-mediated triggering of domain dissociation leads to insertion of SecA into the membrane and activation of its translocase function. Economou's group 69,89 pointed to a specific region near the C-terminus, residues 783-795, that serves as an intramolecular regulator of ATPase activity (IRA1), and proposed that ligands (or deletion) caused the release of this IRA inhibition. They also identified another region that had synergistic effects, and termed it IRA2 and the first segment IRA1.…”

“…IRA1 may be acting as a molecular switch to regulate signal sequence binding, 67 since its deletion also increased binding. 69 The PPXD is implicated in binding to open SecA by the finding that signal peptides stabilize an isolated construct corresponding to the PPXD in a distinct conformation. 89 As expected for a complex interplay of ligand-modulated conformational transitions, the binding of signal peptides shifts the closed to open equilibrium in SecA and modulates its ATPase activity.…”