A nexus of intrinsic dynamics underlies translocase priming

The cytoplasmic ATPase SecA and the membrane-embedded SecYEG channel assemble to form the Sec translocase. How this interaction primes and catalytically activates the translocase remains unclear. We show that priming exploits a nexus of intrinsic dynamics in SecA. Using atomistic simulations, smFRET, and HDX-MS, we reveal multiple dynamic islands that cross-talk with domain and quaternary motions. These dynamic elements are functionally important and conserved. Central to the nexus is a slender stem through which rotation of the preprotein clamp of SecA is biased by ATPase domain motions between open and closed clamping states. An H-bonded framework covering most of SecA enables multi-tier dynamics and conformational alterations with minimal energy input. As a result, cognate ligands select preexisting conformations and alter local dynamics to regulate catalytic activity and clamp motions. These events prime the translocase for high-affinity reception of non-folded preprotein clients. Dynamics nexuses are likely universal and essential in multi-liganded proteins.</p

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated, and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptides promote clamp closing; their mature domain overcomes the rate-limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly “catch and release” trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics, and become secreted

Μοριακή ανάλυση του συστήματος έκκρισης τύπου ΙΙΙ στο εντεροπαθογόνο E.coli

Type III secretion system (T3SS) is a widespread virulence system, in gram negative bacteria, used to transport effector proteins directly into the eukaryotic host cell by spanning three membranes, two bacterial and one eukaryotic. The T3SS is a unique nanomachine that resembles a needle that is used to inject the secretory proteins. The basic structural characteristics of the system are three. The basal body that forms ring-like structures and connects the inner with the outer membrane of the bacterium. From the basal body a needle-filament construction is generated that will be attached to the host cell. In the cytosolic domain of the inner membrane the cytosolic ring and the ATPase complex is assembled so to regulate and coordinate the secretion process. In order the system to become functional and active more than 50 proteins need to be synchronized so the assembly of the system and the protein secretion through it undergo sophisticated regulation. Although recent structural and biochemical studies provide information about the assembly of the system; the understanding of the molecular mechanism behind the translocation process remains elusive.The main goal of this thesis was to shed more light to the molecular mechanism that proteins follow so to be secreted from T3SS. The aim was to investigate and map the pathway proteins follow from the bacterial cytosol towards the membrane at the base of the translocation pore. Determination of the protein-protein interactions that occur and elucidate the mechanism that lies behind the membrane targeting of the secretory protein- chaperone complexes was our ultimate target.Using as a model organism Enteropathogenic E. coli we combined structural, biochemical and biophysical approaches to achieve our goal.Το σύστημα έκκρισης τύπου ΙΙΙ (Τ3SS) είναι ένα ευρεία διαδεδομένο σύστημα που χρησιμοποιείται από πολλά παθογόνα, κατά Gram αρνητικά βακτήρια. Το σύστημα τύπου ΙΙΙ, είναι μια εξειδικευμένη μικρο-μηχανή που χρησιμοποιείται για την μεταφορά των μολυσματικών παραγόντων του βακτηρίου από το κυτταρόπλασμα του κατευθείαν μέσα στο κύταρο ξεωιστή, διαπερνώντας τρεις μεμβρανικές δομές, δυο βακτηριακές και μία του ευκαρυώτη. Το σύστημα έκκρισης τύπου ΙΙΙ σχηματίζει μια δομή στο χώρο που μπορεί να παρομοιαστεί με βελόνα (injectisome). Για την δημιουργία και ενεργοποίηση του συστήματος περίπου 50 πρωτεΐνες πρέπει να συντονιστούν ώστε το σύστημα να απόκτηση τη σωστή διαμόρφωση στο χώρο και οι πρωτεΐνες που πρόκειται να εκκριθούν από αυτό να μεταφερθούν εκεί, η όλοι διαδικασία υπόκειται πολύπλοκο και σύνθετη έλεγχο από διάφορους παράγοντες σε διάφορα επίπεδα κατά το μονοπάτι εξόδου των πρωτεϊνών από το κύτταρο. Παρόλο που πάρα πολλές δομικές και βιοχημικές μελέτες έχουν συμβάλει στην κατανόηση και δομική ανάλυση του συστήματος, ελάχιστες πληροφορίες σχετικά με το μονοπάτι που ακολουθούν οι πρωτεΐνες με στόχο την έξοδό τους από το κύτταρο και την ρύθμιση αυτού είναι γνωστές. Βασικός στόχος της παρούσας διδακτορικής διατριβής είναι η κατανόηση και αποσαφήνιση του μονοπατιού που ακολουθούν οι πρωτεΐνες οι οποίες πρόκειται να εκκριθούν, κατά την μετατόπιση αυτών από το βακτηριακό κυτταρόπλασμα μέχρι την μεμβράνη, στον πόρο εξόδου του συστήματος έκκρισης τύπου ΙΙΙ. Μέλημά μας είναι ο εντοπισμός και χαρακτηρισμός των αλληλεπιδράσεων που συμβαίνουν ανάμεσα στις πρωτεΐνες του συστήματος και η χαρτογράφηση αυτών με στόχο την διασαφήνιση του μηχανισμού που ακολουθείται κατά την στόχευση των πρωτεΐνων στην μεμβράνη

Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system

The flagellum and the injectisome enable bacterial locomotion and pathogenesis, respectively. These nanomachines assemble and function using a type III secretion system (T3SS). Exported proteins are delivered to the export apparatus by dedicated cytoplasmic chaperones for their transport through the membrane. The structural and mechanistic basis of this process is poorly understood. Here we report the structures of two ternary complexes among flagellar chaperones (FliT and FliS), protein substrates (the filament-capping FliD and flagellin FliC), and the export gate platform protein FlhA. The substrates do not interact directly with FlhA; however, they are required to induce a binding-competent conformation to the chaperone that exposes the recognition motif featuring a highly conserved sequence recognized by FlhA. The structural data reveal the recognition signal in a class of T3SS proteins and provide new insight into the assembly of key protein complexes at the export gate.status: publishe

Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase

Bacterial secretory preproteins are translocated across the inner membrane post-translationally by the SecYEG-SecA translocase. Mature domain features and signal peptides maintain preproteins in kinetically trapped, largely soluble, folding intermediates. Some aggregation-prone preproteins require chaperones, like trigger factor (TF) and SecB, for solubility and/or targeting. TF antagonizes the contribution of SecB to secretion by an unknown molecular mechanism. We reconstituted this interaction in vitro and studied targeting and secretion of the model preprotein pro-OmpA. TF and SecB display distinct, unsuspected roles in secretion. Tightly associating TF:pro-OmpA targets the translocase at SecA, but TF prevents pro-OmpA secretion. In solution, SecB binds TF:pro-OmpA with high affinity. At the membrane, when bound to the SecA C-tail, SecB increases TF and TF:pro-OmpA affinities for the translocase and allows pro-OmpA to resume translocation. Our data reveal that TF, a main cytoplasmic folding pathway chaperone, is also a bona fide post-translational secretory chaperone that directly interacts with both SecB and the translocase to mediate regulated protein secretion. Thus, TF links the cytoplasmic folding and secretion chaperone networks.status: publishe

Type III secretion: building and operating a remarkable nanomachine

The Type III secretion system (T3SS) is a protein export pathway that is widespread in Gram-negative bacteria and delivers effector proteins directly into eukaryotic cells. At its core lie the injectisome (a sophisticated transmembrane secretion apparatus) and a complex network of specialized chaperones that target secretory proteins to the antechamber of the injectisome. The assembly of the system, and the subsequent secretion of proteins through it, undergo fine-tuned, hierarchical regulation. Here, we present the current understanding of the injectisome assembly process, secretion hierarchy, and the role of chaperones. We discuss these events in light of available structural and biochemical dissection and propose future directions essential to revealing mechanistic insight into this fascinating nanomachine.publisher: Elsevier articletitle: Type III Secretion: Building and Operating a Remarkable Nanomachine journaltitle: Trends in Biochemical Sciences articlelink: http://dx.doi.org/10.1016/j.tibs.2015.09.005 content_type: article copyright: Copyright © 2015 Elsevier Ltd. All rights reserved.status: publishe

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Förster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.status: publishe

Preprotein conformational dynamics drive bivalent translocase docking and secretion

Most bacterial secretory proteins destined beyond the plasma membrane are secreted post-translationally by the Sec translocase. In the first step of translocation, preproteins are targeted for binding to their 2-site receptor SecA, the peripheral ATPase subunit of the translocase. We now reveal that secretory preproteins use a dual-key mechanism to bridge the signal peptide and mature domain receptor sites and cooperatively enhance their affinities. Docking of targeting-competent mature domains requires that their extensive disorder is finely tuned. This is achieved through amino-terminal mature domain regions acting as conformational rheostats. By being linked to the rheostats, signal peptides regulate long-range preprotein disorder. Concomitant conformational changes in SecA sterically adapt its two receptor sites to optimally recognize hundreds of dissimilar preproteins. This novel intramolecular conformational crosstalk in the preprotein chains and the dynamic interaction with their receptor are mechanistically coupled to preprotein engagement in the translocase and essential for secretion.status: publishe