Targeting SurA Dynamics: An Achilles Heel in Bacterial Outer Membrane Biogenesis

In February 2017 the World Health Organisation published its first ever list of antibiotic-resistant "priority pathogens" - a catalogue of 12 families of bacteria that pose the greatest threat to human health. Of these, 9 are Gram-negative bacteria. The outer membrane (OM) of Gram-negative bacteria protects them from potentially damaging molecules such as antibiotics. The OM is packed with outer membrane proteins (OMPs), creating a permeability barrier that is critical for bacterial survival and endows bacterial resistance to many types of antibiotics. Breaking this barrier is thus an attractive route towards the generation of new antibacterial agents. The biosynthesis of OMPs starts in the cytoplasm and ends with their folding and insertion into the OM, where they form cylindrical, barrel-like structures. The molecular chaperone SurA escorts these newly made unfolded OMPs through the periplasm to the OM. Perturbing SurA chaperone function leads to a loss of bacterial viability and affects antibiotic resistance, demonstrating that SurA is an attractive target to control Gram-negative pathogens.

One of the current challenges of effective manipulation of SurA activity is our very limited understanding of the molecular basis of how this large, multidomain chaperone machine works to ensure the safe journey of OMPs through the periplasm. While the crystal structure of SurA is known, a wealth of evidence suggests that SurA function predominantly relies on dynamic motions in SurA involving interdependent rearrangements of its three domains and their transient interactions with unfolded OMPs. In this proposal we will exploit the very latest, exciting, developments in solution nuclear magnetic resonance (NMR), along with other biophysical and computational techniques and functional assays in test tubes and in living bacteria, that will enable us to 'watch' the dynamic SurA chaperone machinery in action with atomic resolution. Using these experiments we will be able to understand how conformational rearrangements in SurA enable its chaperone function. In addition, we will elucidate how the transient and dynamic interactions between SurA and its OMP substrates enable efficient substrate recognition and prevent OMP aggregation. Finally, we will explore how SurA function and OMP biosynthesis can be controlled in vivo, paving the way towards new antibacterial agents that impair how pathogenic bacteria build their OM, weakening their defences against antibiotics.

SurA is required for the generation of a fully functional OM in Gram negative organisms and hence is a potential target for new anti-bacterial agents. Here we will exploit an "integrative structural biology" approach to determine how SurA conformational dynamics are exploited to enable it to carry out its important functions. Specifically, combining NMR with other biophysical analyses and functional assays in vitro and in vivo, we will:

(i) determine the dynamic conformational landscape of SurA by combining methyl NMR (that allows characterisation of protein systems up to 500kDa) with paramagnetic relaxation enhancement (PRE) and residual dipolar coupling (RDC) measurements to obtain distance restraints, and relaxation dispersion NMR to characterise microsec-millisec conformational transitions and lowly populated (0.5-5%) states. Using all-atom and coarse-grained molecular dynamics we will generate an ensemble of SurA conformations that best describe the experimental data so as to derive a full atomic view of the SurA conformational cycle.

(ii) map the interaction sites between SurA and OMPs and determine how binding alters SurA dynamics. Specifically, we will use a "tag and transfer" cross-linking approach recently developed in SER's laboratory to map the interaction interfaces, and NMR to monitor the effects of binding on SurA dynamics. These experiments will be complemented with H/D exchange (HDX) and smFRET to explore how communication between SurA domains is altered by substrate binding.

(iii) use OMP folding assays in vitro and SurA activity assays in vivo to determine how structural and dynamic changes in SurA (e.g. caused by mutation or peptide binding) affect OMP folding and bacterial viability.

The proposal builds on a wealth of supporting data demonstrating the feasibility of each aspect of the work, despite the challenges of investigating a large and dynamic molecular chaperone that binds unfolded OMPs via transient interactions.

The proposed project will provide mechanistic understanding of how the ATP-independent periplasmic chaperone, SurA, from Gram-negative bacteria assists folding of newly synthesised outer membrane proteins (OMPs) and safely transfers them through the periplasm to be folded and inserted into the outer membrane. Whilst the mechanistic understanding of SurA function is of great biophysical and biomedical interest, and will potentially provide a promising way to specifically target Gram-negative bacteria, little molecular details of SurA action are known due to several fundamental limitations: the transient nature of SurA-OMP interactions, the highly heterogeneous, multistate and dynamic conformational landscape of SurA, the absence of energy sources (ATP) or/and co-chaperones to control and trigger the SurA chaperone cycle, and the large size (60-150 kDa) of SurA-OMP complexes. Our project will address these challenges by exploiting recent developments in nuclear magnetic resonance (NMR) and other biophysical techniques, and by combining these with functional assays in vitro and in vivo.

As Gram-negative bacteria pose major threats to the health and wealth of the UK (9 of the top 12 antibiotic-resistant pathogens are Gram-negative), our results will be of interest to a broad spectrum of life scientists, medicinal chemists and medical researchers in both academia and industry. We will generate a wealth of new information concerning SurA chaperone function and will provide novel insights into how SurA activity can be modulated to perturb OMP biosynthesis, bacterial viability and resistance to antibiotics (see Academic Beneficiaries). SurA represents a particularly attractive, but currently under-explored, route for controlling Gram-negative bacteria, since (i) OMP biogenesis is essential for cell viability; (ii) SurA is conserved in Gram-negative bacteria, but is not present in man; and (iii) small molecules (less than 600 Da) can readily diffuse through the OM. We will also develop an integrated structural biology approach for atomic characterisation of a dynamic multicomponent chaperone system and transient chaperone-substrate interactions that will be of general use and interest by the academic and industrial research communities as a whole for the characterisation of other dynamic protein machines and transient protein-protein interactions. We will make this multidisciplinary experimental approach widely available for future research. In terms of our own research, once the experimental tools have been developed, we intend to exploit them to the full to study other large protein systems of biological and medical interest. Thus, we anticipate opportunities to apply for further grant funding from research councils and charities.

The project outputs and experimental developments will open up a new area of designing small molecules and other potential reagents that specifically target Gram-negative bacteria through the SurA pathway. The results will thus be of interest to the pharmaceutical industry, potentially leading to new and improved strategies to combat antibiotic resistance. More broadly, we expect to develop long-lasting industrial impact through fundamental scientific insights into the molecular mechanisms of the central part of OMP biosynthesis in Gram-negative bacteria. We plan to exploit every opportunity for interacting with relevant industries and other interested parties throughout the programme of study so that we can share results and exploit their potential to the full (see Pathways to Impact).