Allosteric communication between signal peptides and the SecA protein DEAD motor ATPase domain

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.     

SecA-mediated targeting and translocation of secretory proteins

More than 30 years of research have revealed that the dynamic nanomotor SecA is a central player in bacterial protein secretion. SecA associates with the SecYEG channel and transports polypeptides post-translationally to the trans side of the cytoplasmic membrane. It comprises a helicase-like ATPase core coupled to two domains that provide specificity for preprotein translocation. Apart from SecYEG, SecA associates with multiple ligands like ribosomes, nucleotides, lipids, chaperones and preproteins. It exerts its essential contribution in two phases. First, SecA, alone or in concert with chaperones, helps mediate the targeting of the secretory proteins from the ribosome to the membrane. Next, at the membrane it converts chemical energy to mechanical work and translocates preproteins through the SecYEG channel. SecA is a highly dynamic enzyme, it exploits disorder–order kinetics, swiveling and dissociation of domains and dimer to monomer transformations that are tightly coupled with its catalytic function. Preprotein signal sequences and mature domains exploit these dynamics to manipulate the nanomotor and thus achieve their export at the expense of metabolic energy. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Coiled-coils in type III secretion systems: structural flexibility, disorder and biological implications

Recent structural studies and analyses of microbial genomes have consolidated the understanding of the structural and functional versatility of coiled-coil domains in proteins from bacterial type III secretion systems (T3SS). Such domains consist of two or more α-helices forming a bundle structure. The occurrence of coiled-coils in T3SS is considerably higher than the average predicted occurrence in prokaryotic proteomes. T3SS proteins comprising coiled-coil domains are frequently characterized by an increased structural flexibility, which may vary from localized structural disorder to the establishment of molten globule-like state. The propensity for coiled-coil formation and structural disorder are frequently essential requirements for various T3SS functions, including the establishment of protein–protein interaction networks and the polymerization of extracellular components of T3SS appendages. Possible correlations between the frequently observed N-terminal structural disorder of effectors and the T3SS secretion signal are discussed. The results for T3SS are also compared with other Gram-negative secretory systems.