Assembly of the translocase motor onto the preprotein-conducting channel.

Bacterial protein secretion is catalysed by the SecYEG protein-conducting channel complexed with the SecA ATPase motor. To gain insight into the SecA-SecYEG interaction we used peptide arrays, thermodynamic quantification, mutagenesis and functional assays. Our data reveal that: (i) SecA binds with low affinity on several, peripheral, exposed SecYEG sites. This largely electrostatic association is modulated by temperature and nucleotides. (ii) Binding sites cluster in five major binding 'regions': three that are exclusively cytoplasmic and two that reach the periplasm. (iii) Both the N-terminal and c-terminal regions of SecA participate in binding interactions and share some sites. (iv) Several of these sites are essential for translocase catalysis. Our data provide residue-level dissection of the SecYEG-SecA interaction. Two models of assembly of SecA on dimeric SecYEG are discussed.

Preprotein-controlled catalysis in the helicase motor of SecA.

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.

Identification of the preprotein binding domain of SecA.

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.

Purification of a functional mature region from a SecA-dependent preprotein.

Most of the bacterial proteins that are active in extracytoplasmic locations are translocated through the inner membrane by the Sec translocase. Translocase comprises a membrane "pore" and the peripheral ATPase SecA. Where preproteins bind to SecA and how they activate translocation ATPase remains elusive. To address this central question we have purified to homogeneity the mature and preprotein parts of an exported protein (pCH5EE). pCH5EE satisfies a minimal size required for protein translocation and its membrane insertion is SecA-dependent. Purified pCH5EE and CH5EE can form physical complexes with SecA and can functionally suppress the elevated ATPase of a constitutively activated mutant. These properties render pCH5EE and CH5EE unique tools for the biochemical mapping of the preprotein binding site on SecA.

Escherichia coli SecA truncated at its termini is functional and dimeric.

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase.

The SecA ATPase is a protein translocase motor and a superfamily 2 (SF2) RNA helicase. The ATPase catalytic core ('DEAD motor') contains the seven conserved SF2 motifs. Here, we demonstrate that Motif III is essential for SecA-mediated protein translocation and viability. SecA Motif III mutants can bind ligands (nucleotide, the SecYEG translocase 'channel', signal and mature preprotein domains), can catalyse basal and SecYEG-stimulated ATP hydrolysis and can be activated for catalysis. However, Motif III mutation specifically blocks the preprotein-stimulated 'translocation ATPase' at a step of the reaction pathway that lies downstream of ligand binding. A functional Motif III is required for optimal ligand-driven conformational changes and kinetic parameters that underlie optimal preprotein-modulated nucleotide cycling at the SecA DEAD motor. We propose that helicase Motif III couples preprotein binding to the SecA translocation ATPase and that catalytic activation of SF2 enzymes through Motif-III-mediated action is essential for both polypeptide and nucleic-acid substrates.