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 conformational space sampled by the two-domain protein calmodulin has been explored by an approach based on four sets of NMR observables obtained on Tb 3؉ -and Tm 3؉ -substituted proteins. The observables are the pseudocontact shifts and residual dipolar couplings of the C-terminal domain when lanthanide substitution is at the N-terminal domain. Each set of observables provides independent information on the conformations experienced by the molecule. It is found that not all sterically allowed conformations are equally populated. Taking the N-terminal domain as the reference, the C-terminal domain preferentially resides in a region of space inscribed in a wide elliptical cone. The axis of the cone is tilted by Ϸ30°with respect to the direction of the N-terminal part of the interdomain helix, which is known to have a flexible central part in solution. The C-terminal domain also undergoes rotation about the axis defined by the C-terminal part of the interdomain helix. Neither the extended helix conformation initially observed in the solid state for free calcium calmodulin nor the closed conformation(s) adopted by calcium calmodulin either alone or in its adduct(s) with target peptide(s) is among the most preferred ones. These findings are unique, both in terms of structural information obtained on a biomolecule that samples multiple conformations and in terms of the approach developed to achieve the results. The same approach is in principle applicable to other multidomain proteins, as well as to multiple interaction modes between two macromolecular partners. C almodulin (CaM) is a paradigm case in structural biology. The following brief survey of the history of the structural and dynamic studies on this protein serves the double purpose of putting the present findings in proper perspective and of acknowledging those pieces of previous information that were essential to allow the present approach to be developed and to yield novel structural information.CaMs are two-domain proteins belonging to the large family of EF-hand proteins (1-3). They contain Ϸ150 amino acid residues, organized into two domains of Ϸ70 aa each and connected by a short linker. Each domain is made up of two special helix-loop-helix motifs (EF-hand motifs) that can bind a calcium ion in the loop. The two loops are held close to one another by two short antiparallel ␤-strands forming a threehydrogen bond stretch of ␤-sheet. The function of CaM in cell cytoplasm is that of responding to sudden rises of calcium concentration by binding up to four calcium ions in the four EF-hand loops, by changing conformation because of metal binding, and by thus becoming able to recognize, bind to, and activate, a number of proteins and enzymes (1,(4)(5)(6)(7)(8). Early x-ray data (9) showed the four-calcium (Ca 2 ) N (Ca 2 ) C CaM form (subscripts N and C refer to the calcium atoms bound by the N-and C-terminal domains, respectively) to have a dumbbell shape, with helix 4, the last helix of the N-terminal domain, and helix 5, the first helix of the C-termi...

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 ,