SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase. SecA comprises a dimerization C-terminal domain fused to an ATPase N-terminal domain containing conserved DEAD helicase motifs. We show that the N-terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2. NBD1, a rigid nucleotide-binding domain, contains the minimal ATPase catalytic machinery. IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controling ADP release and optimal ATP catalysis at NBD1. IRA2 is¯exible and can undergo changes in its a-helical content. The C-terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function. Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP-bound state and unnecessary ATP hydrolysis cycles are prevented. Two DEAD family motifs in IRA2 are essential for IRA2±NBD1 binding, optimal nucleotide turnover and polypeptide translocation. We propose that translocation ligands alleviate C-terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1. DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.
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 ...
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 crosstalk 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.
The cornerstone of the functionality of almost all motor proteins is the regulation of their activity by binding interactions with their respective substrates. In most cases, the underlying mechanism of this regulation remains unknown. Here, we reveal a novel mechanism used by secretory preproteins to control the catalytic cycle of the helicase ‘DEAD' motor of SecA, the preprotein translocase ATPase. The central feature of this mechanism is a highly conserved salt‐bridge, Gate1, that controls the opening/closure of the nucleotide cleft. Gate1 regulates the propagation of binding signal generated at the Preprotein Binding Domain to the nucleotide cleft, thus allowing the physical coupling of preprotein binding and release to the ATPase cycle. This relay mechanism is at play only after SecA has been previously ‘primed’ by binding to SecYEG, the transmembrane protein‐conducting channel. The Gate1‐controlled relay mechanism is essential for protein translocase catalysis and may be common in helicase motors.
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