Two Partner Secretion in bacteria: Conformational dynamics of the FhaC transporter. – DYN-FHAC

FhaC forms a channel in the outer membrane preceded by periplasmic domains. To study the conformational changes leading to channel opening, we are using biophysical approaches that provide the structure of FhaC in solution and the mobility of its structural elements. We also analyse how FHA and FhaC interact in the transport process. Finally we are characterizing the X ray structures of homologue of these two proteins.

Our multidisciplinary approaches at several levels of analysis (with the purified protein, in bacteria and in silico) have led to the discovery that the transporter FhaC is highly mobile, especially in a membrane environment. FhaC goes through important conformation changes when it transports FHA across the membrane.

Our next objective is to use a sophisticated biophysical method called pulsed electron paramagnetic resonance to measure distances within FhaC in order to confirm and refine our current results. We will combine this approach with in silico molecular modelling thanks to the collaboration of a specialist who recently joined our consortium.

In 2011 we published two articles on this system, one describing the recognition between FHA and FhaC and the other the role of a molecular chaperone called DegP in the TPS pathway. In collaboration with a German group, we published in 2012 an article reporting the reconstitution of the transport of FHA by FhaC in vitro. In collaboration with a Belgian group we have submitted for publication our work on the folding of FHA during its

Pathogenic bacteria have evolved mechanisms to secrete proteins such as enzymes, toxins and adhesins that enable them to interact efficiently with the host environment and to perform their infectious cycle. In Gram-negative bacteria the presence of an outer membrane has required the development of specific systems to address these proteins to their proper location. Understanding the molecular mechanisms of these secretion systems which represent an essential aspect of bacterial pathogenesis is important from a basic science perspective, and also because these systems are potentially new therapeutic targets.
The Two-Partner Secretion (TPS) pathway is widespread in Gram-negative bacteria, including important pathogens such as Bordetella pertussis, Neisseria meningitidis or Pseudomonas aeruginosa, and it is devoted to the secretion of large adhesins and cytolysins that fold into long beta helices. The two partners of these systems are the secreted TpsA protein harbouring a conserved, N-terminal TPS domain essential for secretion, and its specific TpsB transporter forming a pore in the outer membrane. The secretion system of the FHA adhesin of B. pertussis by its FhaC partner serves as a model for the TPS pathway. FHA (TpsA partner) is an important virulence factor of this pathogen. FhaC (TpsB partner) belongs to the Omp85/TpsB superfamily which includes notably essential protein transporters of mitochondria and chloroplasts. Our system thus represents also a model to decipher the molecular mechanisms in this superfamily.
We obtained in 2007 the X ray structure of FhaC, which has remained thus far the only one of the TpsB/Omp85 superfamily. FhaC forms a 16-stranded transmembrane beta barrel preceded by a periplasmic domain composed of two POTRA domains. Large extracellular loops and short periplasmic turns connect the anti-parallel strands of the barrel. The barrel pore is obstructed by a long N-terminal alpha helix (H1) and an extracellular loop (L6) folded back into the barrel. H1 is followed by a periplasmic linker that connects it to the first POTRA. We have demonstrated the functional role of the two POTRAs for the molecular recognition of the TPS domain of FHA by FhaC and the role of L6 and the linker for the activity of FhaC. Our current model of secretion based on numerous experimental results goes as follows. Following Sec-dependent export of FHA through the cytoplasmic membrane, the TPS domain of FHA in an extended conformation interacts with the POTRAs. This triggers the opening of the pore, leading to the progressive translocation of FHA in an extended conformation through the FhaC pore. FHA folds progressively at the cell surface to form its extended beta helix. The X ray structure of FhaC most likely represent the resting state of the transporter, which will undergo significant conformational changes during the secretion cycle, such as the move of H1, L6 or both out of the pore.
The proposed program rests on the structure of FhaC and our experience on this model system. Our goal is to decipher the conformational dynamics of FhaC in the secretion cycle at the molecular level. We will combine X ray crystallography, structural approaches in solution (SANS), biochemical and biophysical methods such as cross-linking in vivo and electronic paramagnetic resonance, to address the conformational changes of FhaC in the course of secretion and to obtain the structure of FhaC in action. In particular, we will analyse the dynamics of important structural elements (POTRAs, H1, linker, L6, other surface loops) and compare them between the resting and active states. We will define the pathway followed by FHA through its transporter by probing its interactions with these various elements. We want to obtain the structure of FhaC in action, by preparing a blocked complex between FhaC and a chimeric protein containing a secretion-competent N-terminal FHA fragment fused to a heterologous, secretion-incompetent globular domain.