No abstract

Recognition of signal sequences by cognate receptors controls the entry of virtually all proteins to export pathways. Despite its importance, this process remains poorly understood. Here, we present the solution structure of a signal peptide bound to SecA, the 204 kDa ATPase motor of the Sec translocase. Upon encounter, the signal peptide forms an alpha-helix that inserts into a flexible and elongated groove in SecA. The mode of binding is bimodal, with both hydrophobic and electrostatic interactions mediating recognition. The same groove is used by SecA to recognize a diverse set of signal sequences. Impairment of the signal-peptide binding to SecA results in significant translocation defects. The C-terminal tail of SecA occludes the groove and inhibits signal-peptide binding, but autoinhibition is relieved by the SecB chaperone. Finally, it is shown that SecA interconverts between two conformations in solution, suggesting a simple mechanism for polypeptide translocation.

The SecA subunit of E. coli preprotein translocase promotes protein secretion during cycles of membrane insertion and deinsertion at SecYEG. This process is regulated both by nucleotide binding and hydrolysis and by the SecD and SecF proteins. In the presence of associated preprotein, the energy of ATP binding at nucleotide-binding domain 1 (NBD1) drives membrane insertion of a 30 kDa domain of SecA, while deinsertion of SecA requires the hydrolysis of this ATP. SecD and SecF stabilize the inserted state of SecA. ATP binding at NBD2, though needed for preprotein translocation, is not needed for SecA insertion or deinsertion.

The general secretory (Sec) pathway comprises an essential, ubiquitous and universal export machinery for most proteins that integrate into, or translocate through, the plasma membrane. Sec exportome polypeptides are synthesized as pre-proteins that have cleavable signal peptides fused to the exported mature domains. Recent advances have re-evaluated the interaction networks of pre-proteins with chaperones that are involved in pre-protein targeting from the ribosome to the SecYEG channel and have identified conformational signals as checkpoints for high-fidelity targeting and translocation. The recent structural and mechanistic insights into the channel and its ATPase motor SecA are important steps towards the elucidation of the allosteric crosstalk that mediates secretion. In this Review, we discuss recent biochemical, structural and mechanistic insights into the consecutive steps of the Sec pathway - sorting and targeting, translocation and release - in both co-translational and post-translational modes of export. The architecture and conformational dynamics of the SecYEG channel and its regulation by ribosomes, SecA and pre-proteins are highlighted. Moreover, we present conceptual models of the mechanisms and energetics of the Sec-pathway dependent secretion process in bacteria.

Molecular chaperones prevent aggregation and misfolding of proteins but scarcity of structural data has impeded an understanding of the recognition and anti-aggregation mechanisms. Here we report the solution structure, dynamics and energetics of three Trigger Factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. NMR relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion but as the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and how by acting as unfoldases and holdases prevent the aggregation and premature (mis)folding of unfolded proteins.

No abstract

Extra-cytoplasmic polypeptides are usually synthesized as "preproteins" carrying aminoterminal, cleavable signal peptides 1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA 2,3 . Most proteins destined for the periplasm and beyond are exported post-translationally by SecA 2,3 . Preprotein targeting to SecA is thought to involve signal peptides 4 and chaperones like SecB 5,6 . Here we reveal that signal peptides have a novel role beyond targeting: they are essential allosteric activators of the translocase. Upon docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, "triggering" that drives the translocase to a lower activation energy state; then "trapping" that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus and, finally, "secretion" during which trapped mature domains undergo multiple turnovers of translocation in segments 7 . A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases.We sought to dissect the individual contributions of signal peptides and mature domains to membrane targeting and to post-targeting translocation steps. Since SecB is not universal or essential 6,8 , we used the SecB-independent 9,10 substrate proPhoA (periplasmic alkaline phosphatase).The affinity of proPhoA for inverted inner membrane vesicles (IMVs) containing SecYEG either alone or complexed with SecA was determined (Fig. 1a). ProPhoA associates with high affinity (0.23 μM) to SecYEG-bound SecA but not to SecYEG alone. Like proOmpA 5 ,

All cells must traffic proteins across their membranes. This essential process is responsible for the biogenesis of membranes and cell walls, motility and nutrient scavenging and uptake, and is also involved in pathogenesis and symbiosis. The translocase is an impressively dynamic nanomachine that is the central component which catalyses transmembrane crossing. This complex, multi-stage reaction involves a cascade of inter- and intramolecular interactions that select, sort and target polypeptides to the membrane, and use energy to promote the movement of these polypeptides across--or their lateral escape and integration into--the phospholipid bilayer, with high fidelity and efficiency. Here, we review the most recent data on the structure and function of the translocase nanomachine.