Does functional misfolding of TonB drive import across the outer membrane of Gram negative bacteria?

Proteins carry out a wide-range of important functions that are essential for life, including the acquisition and metabolism of vital nutrients that are scarce in the environment. It is well accepted that, to be functional, most proteins need to fold to well a defined and stable three dimensional shape, known as their structure or conformation. More recently, however, proteins with no fixed structure or regions of no fixed structure called intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) have emerged. These proteins play an important role in signalling by both responding to changes in the environment and by their ability to interact with a multitude of other proteins and other complex cellular molecules. As these proteins don't have a fixed structure and are very sensitive to even small changes in the environment, they are challenging to study and despite their importance, the precise way in which work is still being investigated. We have been studying a protein called TonB which contains an IDR that was thought to act merely as a passive linker between the two active parts of the protein at each end of this elongated protein. By stretching single protein molecules using an atomic force microscope we found that under some conditions this supposedly unstructured protein was able to resist extension (akin to a slip knot in a rope). The ability of TonB to toggle between a force-resistant, structured conformation and an unfolded conformation by changes in environment is completely novel and understanding how this works may help to understand a long standing question of how bacteria acquire some nutrients.

The import of nutrients is problematic for a large class of bacteria (Gram negative) which have a protective barrier outside their cell membrane called the outer membrane. While allowing their survival in harsh conditions, the outer membrane also acts as a barrier to the import of large nutrients. As there is no energy source in the space between outer and inner membrane (called the periplasm), any process requiring energy, such as the import of scarce nutrients, has to be driven from an energy source at the inner membrane. TonB carries out this inside-out energy transduction by altering the structure of import proteins located in the outer membrane but its mechanism is unknown. The aim of this research is to understand how changes in the structure and dynamics of the IDR of TonB that spans the periplasm drives import. This is important as a molecular-level understanding this mechanism would both reveal a novel function for IDP/Rs in cellular signalling and act as starting point for the design of novel anti-bacterial agents.

The presence of an impermeability barrier (the outer membrane) distal from the energised inner membrane is essential for survival but poses several problems to Gram negative bacteria exemplified by the import of vital but scarce nutrients such as vitamin B12. While porins in the OM allow passive diffusion of relatively small molecules (<600 Da), nutrients such as vitamin B12 are both larger and more scarce, obviating passive diffusion as a method to their acquirement (and enrichment) in vivo. Instead, an array of specialist OM proteins called TonB dependent transporters (TBDT) each bind specific substrates with high affinity and transport them through lumen that are gated by plug domains. How the plug domains are re-structured ('uncorked' or remodelled) to allow substrate transit to the periplasm is still unknown but the process requires a functional ExbB/D:TonB complex. TonB comprises at least three distinct regions: an N-terminal transmembrane domain that resides in the energised inner membrane, a C-terminal globular domain that binds to the TBDT plug domain with these regions linked by a largely unstructured region, rich in proline and charged residues. Whilst it is clear that an energised inner membrane (i.e. the presence of the proton motive force) is required for its function, the mechanism by which TonB drives TBDT plug remodelling is not known.

Our group has previously shown that the disordered linker region of TonB displays properties that are unusual for an intrinsically disordered protein (IDP). Analysis of the end-to-end lengths of TonB by force spectroscopy revealed two distinct conformations (a compact, force resistant conformation and an extensible conformation) whose relative population is determined by ionic strength. The aim of this research is to understand how TonB toggles between these distinct states, the molecular basis of mechanical strength within this IDP and how this is used in vivo to drive TBDT plug remodelling.