Fonctionally relevant conformations of the protein transporter FhaC: probing the lateral opening of a beta barrel in a biological membrane – OPEN_BAR

The Two-Partner Secretion (TPS) pathway is dedicated to the export of large proteins notably serving as virulence factors. The TpsB transporters are transmembrane ß-barrel proteins that secrete their TpsA substrates across the outer membrane of various Gram-negative bacterial pathogens. They belong to the ubiquitous Omp85 superfamily, whose members mediate protein insertion into, or translocation across, membranes of bacteria and of eukaryotic organelles, and which also includes the bacterial BamA transporters. We are interested in FhaC, the TpsB protein that mediates secretion of the FHA adhesin in Bordetella pertussis. The FHA/FhaC pair is a model TPS system, and FhaC is a recognized Omp85 paradigm. The Omp85 transporters perform their functions in the absence of ATP or an electrochemical gradient. From a basic-science perspective, how they mediate protein transport is a focus of intense interest. From an applied perspective, BamA and TpsB proteins are new potential antibacterial targets.
Omp85 transporters are composed of N-terminal intracellular POTRA domains and a C-terminal transmembrane ß barrel, which in FhaC is the FHA translocation pore. The POTRA domains mediate protein-protein interactions, and notably substrate recognition in the periplasm. A hallmark Omp85 feature is the conserved extracellular loop L6 whose tip interacts with a conserved motif in the inner ß-barrel wall. Additionally, in FhaC, an N-terminal helix called H1 plugs the ß barrel. Both H1 and L6 stabilize the barrel in a closed, resting conformation whose crystal structure is known. The ß barrel, L6 and the last POTRA domain are all essential for function. The molecular mechanisms of action of FhaC remain to be characterized, and this is the goal of this proposal.
FhaC is very dynamic, and the spontaneous exit of H1 from the barrel is one of the conformational changes linked to its transport activity. Our hypothesis is that secretion occurs by a cyclic mechanism involving an enlargement of the FhaC barrel. Entry of the substrate protein into the pore and its progression towards the cell surface are accomplished by reversible conformational changes of FhaC that involve a lateral opening of its beta barrel and the insertion of a portion of the POTRA2 domain into the resulting gap. This change of conformation hoists the substrate bound to POTRA2 from the periplasm into the barrel. The substrate is released towards the cell surface, and the POTRA domains switch back to the periplasm to repeat the cycle. Conformational changes of the L6 loop may stabilize alternative barrel conformations or prevent the substrate from backtracking in the pore.

This cycle implies alternative, transient conformations of FhaC that we will explore using approaches suitable to detect minor states in heterogeneous populations and to track protein dynamics at different levels of resolution. We will combine nuclear magnetic resonance spectroscopy, ion mobility mass spectrometry, hydrogen-deuterium exchange mass spectrometry and crystallography to characterize wild type FhaC in detergent and lipid media. A small set of variants engineered by mutagenesis either to freeze the protein in a closed conformation or to displace the conformational equilibrium of the transporter towards its open form will be selected. The variants will be analyzed in detergent medium and in membrane-mimic environments, and their conformations will be compared using the same set of biophysical techniques. We will determine the energy landscape and the rates of the conformational changes occurring in the secretion cycle. We will also try to solve the structure of alternative forms of FhaC by X-ray crystallography with the help of nanobodies. Combining information from these complementary approaches will enable us to propose a model for the function of FhaC, which we expect to describe a new paradigm for protein transport across a biological membrane.