Molecular basis of protein translocation through outer membrane porins

Gram-negative bacteria are surrounded by two membranes, the outer most of which (usually referred to as the 'outer membrane') is a highly effective barrier against toxic molecules; for example, bile salts which exist within the gut of a mammal. The effectiveness of the outer membrane is a double-edged sword for the organism since it is also very effective at keeping out essential nutrients such as sugars, which the organism needs to grow and divide. Hence all organisms that have an outer membrane also have specialised proteins within this membrane whose job it is to allow the exchange of nutrients and metabolites with the environment. These proteins are known as porins, which are barrel-like membrane proteins that have a hole or pore running through them which traverses the membrane. There are many barrel proteins in the outer membrane of Gram-negative bacteria (it is estimated that 2-3% of the E. coli genome encode such proteins) which serve a variety of functions. Those that allow the exchange of nutrients are usually referred to as general or classical porins, the best understood of which are the proteins OmpF and OmpC. These porins are also the major routes by which commonly used antibiotics (e.g. ampicillin) diffuse into the cell. Indeed, the channels of these porins are frequently found to be mutated in multidrug resistant bacteria.

The focus of this research proposal is a novel function we have recently discovered for the general porin OmpF which shows that porins can do more than just let small molecules diffuse through their pores. We have discovered that OmpF can also be used to allow proteins to pass (translocate) into the cell, as long as the protein is unfolded (i.e. a random coil) and can snake its way through the narrow pores of the porin. Even more remarkably, this translocating protein snakes through two of the three pores of OmpF, which is composed of three barrel subunits; i.e. it goes into the cell then comes back out again. Given the importance of porins to the physiology of bacteria and eukaryotes (mammals and plants have porins in the outer membranes of some of their organelles) our discovery has important ramifications for our understanding of the biology of organisms that have outer membranes since it shows that proteins can exploit their porins. Proteins have functions which are usually imparted by their having a three dimensional structure or fold. Although the proteins that pass through OmpF are unfolded polypeptides and so by implication have no function, these can become functional by virtue of their being able to bind to other molecules such as proteins, and hence in this way alter cellular behaviour. The twin aims of this proposal are to understand the molecular basis for protein translocation through the porin OmpF in the gut bacterium Escherichia coli (determining for example how much polypeptide can pass through the pore) and to discover how common this phenomenon is by looking at other porins and porins from other organisms. Another important question to be investigated is what drives protein translocation through porins? This is an important question since the outer membrane has no energy source to call upon in order to drag a protein into a cell (unlike the inner most membrane which does) and so answering this question will not only provide us with fundamental new insights into bacterial porins themselves but also the outer membrane in which they reside.

We have discovered that an intrinsically disordered protein (IDP) domain of a colicin can thread its way through two subunits of the trimeric porin OmpF in order to deliver an epitope signal to the bacterial periplasm. This proposal aims to uncover the molecular mechanism of this new form of protein translocation and determine if it occurs in other systems.

(1) How does an IDP associate with a porin and what is the driving force for translocation?
This will be investigated by crystallographic, mutational, thermodynamic (calorimetry), and kinetic studies (stopped-flow FRET and planar lipid bilayer (PLB) single channel recordings), all ultimately feeding into molecular dynamics simulations of IDP binding and translocation through porins.

(2) How does an IDP thread through OmpF and how much sequence can be translocated?
The threading mechanism will be investigated using constructs of colicin E9 (1-62) that contain both its OmpF binding sites and its intervening signalling epitope in PLB experiments. We will also determine what constraints there are in the amount of polypeptide that can be translocated.

(3) What is the relationship between IDP sequence and porin binding/translocation?
We will identify OmpF binding sites within the many colicins that are known to utilise OmpF for their import to determine what governs their binding to porins, exploiting a newly devised native state electrospray ionisation mass spectrometry method we have developed.

(4) Do porins other than OmpF bind and translocate IDPs?
We will investigate IDP binding/translocation in other porins, principally OmpC and PhoE to determine differences/similarities with OmpF, and identify/study porins from the PDB with the potential translocate IDPs.

(5) Can a folded domain be delivered through OmpF if tethered to a colicin's IDP?
We will determine if a small, folded domain present in a colicin known to translocate its preceding IDP through OmpF can also be unfolded/translocated.

Who might benefit from this research (excluding other academics) and how might they benefit?

The main beneficiaries of this research will be:

(i) the biotechnology industry, especially those with interests in the use of protein pores in analytical applications (biosensors, protein sequencing). As part of our Impact plan we will engage with scientists at Oxford Nanopore Technologies to discuss potential ways to exploit our discoveries in a way that dovetails with their priorities. Hagan Bayley was one of the co-founders of ONT, which has raised over £100M in venture capital. As part of our research programme we will discover how IDPs interact with/translocate through different porins, which could be valuable in the development of novel biosensors. Finding novel ways of delivering proteins and peptides into cells could have far reaching consequences for biological research and for biotechnology. We will uncover the constraints to the translocation mechanism through porins. We will uncover the ground rules for porin translocation and so be able to design peptide modules, for example, that could bind periplasmic proteins while carrying defined fluorophores. Porin translocation might also be a route by which epitopes could be displayed from the surfaces of bacteria, for example in response to an extracellular signal. Through the University's Technology Transfer Office (Isis Innovation) we will explore the possibility of patenting a novel bacterial surface display technology we are in the process of developing, which is based on IDP translocation through porins and the presentation of epitopes on the cell surface. We will also make links with companies that develop antimicrobial peptides, some of which likely use porins for entry into Gram-negative bacteria. Our research on IDP translocation through porins may provide avenues for the production of more efficient antimicrobial peptides. Protein secretion through bacterial porins has recently been described in the literature, which must almost certainly pass through porins as IDPs. Our work on IDP translocation will provide possible mechanisms for such transfer. The secretion of proteins to the extracellular medium is an important goal in biotechnology for the facile production of proteins (for use in analytical applications or as biologics).

(ii) the general public. A range of outreach and research dissemination activities are planned. The main objectives are to, first, explain the importance of this research for understanding how antimicrobial proteins enter bacteria as this may be a means of developing novel antibacterial strategies (although this is not an objective of this specific proposal), and second, using this as a springboard for explaining concepts as 'what an Intrinsically Disordered Protein (IDP) is?' and 'how protein antibiotics differ from traditional antibiotics'. These educational goals are important as part of a wider objective here in Oxford to prepare the public for new forms of antibiotics that include proteins. A variety of avenues will be used to conduct this outreach. Including Science Open day events organised through the Departments of Biochemistry and Chemistry, public talks (CK to give a secondary school lecture organised by the Biochemical Society), production of non-specialist websites (e.g. Proteopedia) that describe IDPs and in particular how they can pass through narrow porin pores and disseminating web material suitable for a lay audience to BBSRC (e.g. as was recently done for our Science paper which underpins this application; http://www.bbsrc.ac.uk/news/ health/2013/130703-n-antibiotics-sneak-across-membranes.aspx)