Molecular grammar of SurA-client interactions in the periplasm of gram-negative bacteria
Lead Research Organisation:
University of Leeds
Department Name: Sch of Molecular & Cellular Biology
Abstract
Antibiotic resistance is projected to cause 10 million deaths per year by 2050, with gram-negative pathogens comprising 9 of the 12 bacteria that pose the greatest threat to human health, according to the World Health Organisation. These gram-negative pathogens have a unique outer membrane (OM) that acts as a first line of defence against an assault from potentially harmful molecules to the bacteria, such as antibiotics. As a result, the OM is essential for bacterial survival and is one reason why certain bacteria are resistant to different types of antibiotics. Finding ways to prevent correct assembly of the OM may therefore produce new routes to kill gram-negative bacteria, or make them more susceptible to existing antibiotics. However, the mechanism by with the OM is built remains mysterious, making its assembly difficult to target with therapeutics.
Here we propose to determine how key proteins of the OM - so-called outer membrane proteins (or OMPs) - are folded into the OM to create the usually impenetrable cell wall. OMPs play essential roles in bacterial virulence and survival, so by understanding how OMPs are assembled into the OM it may be possible to develop new drugs that target this essential process. A key protein involved in ensuring OMPs reach the OM is a chaperone protein called SurA. SurA is an attractive target for the development of new drugs to control gram-negative pathogens because perturbing the chaperone function of SurA results in a loss of bacterial viability and virulence along with increased sensitivity to antibiotics. However, in order to target the chaperone function of SurA, further work is needed to understand its mechanism of action. We have recently discovered that two key sites on SurA are responsible for recognising OMPs. This is exciting, as it suggests that one or both of these sites could make good targets for new drug-like molecules. However, we still do not understand how each of these two binding sites contribute to OMP binding and chaperone function. Here we propose to use information from an array of complementary and cutting-edge experimental methodologies (including NMR spectroscopy, mass spectrometry, single molecular Forster resonance energy transfer, biochemistry/biophysics and bioinformatics) to understand how each of the newly discovered OMP binding sites on SurA recognises specific signals within its OMP clients. Further, we propose to determine how these two binding sites work together to bring about its chaperone function, in particular regarding SurA's role in protecting newly synthesised OMPs from aggregation and facilitating their delivery to the OM. This will uncover the molecular features of SurA that are essential for assisting in OMP biogenesis, which, in the future, could lead to new strategies to develop much-needed antibiotics that target gram-negative pathogens that threaten humans, plants and animals.
Here we propose to determine how key proteins of the OM - so-called outer membrane proteins (or OMPs) - are folded into the OM to create the usually impenetrable cell wall. OMPs play essential roles in bacterial virulence and survival, so by understanding how OMPs are assembled into the OM it may be possible to develop new drugs that target this essential process. A key protein involved in ensuring OMPs reach the OM is a chaperone protein called SurA. SurA is an attractive target for the development of new drugs to control gram-negative pathogens because perturbing the chaperone function of SurA results in a loss of bacterial viability and virulence along with increased sensitivity to antibiotics. However, in order to target the chaperone function of SurA, further work is needed to understand its mechanism of action. We have recently discovered that two key sites on SurA are responsible for recognising OMPs. This is exciting, as it suggests that one or both of these sites could make good targets for new drug-like molecules. However, we still do not understand how each of these two binding sites contribute to OMP binding and chaperone function. Here we propose to use information from an array of complementary and cutting-edge experimental methodologies (including NMR spectroscopy, mass spectrometry, single molecular Forster resonance energy transfer, biochemistry/biophysics and bioinformatics) to understand how each of the newly discovered OMP binding sites on SurA recognises specific signals within its OMP clients. Further, we propose to determine how these two binding sites work together to bring about its chaperone function, in particular regarding SurA's role in protecting newly synthesised OMPs from aggregation and facilitating their delivery to the OM. This will uncover the molecular features of SurA that are essential for assisting in OMP biogenesis, which, in the future, could lead to new strategies to develop much-needed antibiotics that target gram-negative pathogens that threaten humans, plants and animals.
Technical Summary
Beta-barrel outer membrane proteins (OMPs) perform many essential functions involved in bacterial viability, virulence and survival. SurA is the primary chaperone of OMPs in the periplasm of gram-negative bacteria, but the mechanism of SurA function remains elusive. In particular, it remains unclear how SurA recognises OMPs, how it prevents aggregation, and how it delivers OMPs to the beta-barrel assembly machinery (BAM) complex, which folds and inserts OMPs into the OM.
Here we propose to exploit recent, exciting developments in our laboratories in which we have unexpectedly discovered that SurA contains two specific OMP binding sites, and that SurA binding modulates OMP conformational dynamics when chaperone bound. This suggests a new mechanism for SurA function that relies on coordination of the two binding sites to disfavour the aggregation of a bound client and ready it for delivery to BAM. Using a combination of methyl TROSY NMR, single molecule FRET, MS, peptide arrays and biochemical/biophysical analysis of binding and OMP folding, and combining studies on specific OMPs with E. coli proteome-wide assays, we propose to address the following questions:
1. What is the molecular grammar of the SurA:OMP interaction? Are there sequence-motifs within OMPs that are specifically recognised by each of the binding sites on SurA?
2. How do each of the two binding sites on SurA contribute to altering the conformational dynamics of its bound OMPs, poising them for folding by BAM and preventing their aggregation?
3. Do the two OMP binding sites on SurA play essential roles in the folding of specific OMP clients in E. coli?
The outcome will be a new understanding of the molecular mechanism of a chaperone that is involved in the biogenesis of the OM. In the longer-term the discoveries made could inform new strategies to impair OMP recognition by SurA, with potential implications for the development of new anti-bacterial therapies that target OM biogenes
Here we propose to exploit recent, exciting developments in our laboratories in which we have unexpectedly discovered that SurA contains two specific OMP binding sites, and that SurA binding modulates OMP conformational dynamics when chaperone bound. This suggests a new mechanism for SurA function that relies on coordination of the two binding sites to disfavour the aggregation of a bound client and ready it for delivery to BAM. Using a combination of methyl TROSY NMR, single molecule FRET, MS, peptide arrays and biochemical/biophysical analysis of binding and OMP folding, and combining studies on specific OMPs with E. coli proteome-wide assays, we propose to address the following questions:
1. What is the molecular grammar of the SurA:OMP interaction? Are there sequence-motifs within OMPs that are specifically recognised by each of the binding sites on SurA?
2. How do each of the two binding sites on SurA contribute to altering the conformational dynamics of its bound OMPs, poising them for folding by BAM and preventing their aggregation?
3. Do the two OMP binding sites on SurA play essential roles in the folding of specific OMP clients in E. coli?
The outcome will be a new understanding of the molecular mechanism of a chaperone that is involved in the biogenesis of the OM. In the longer-term the discoveries made could inform new strategies to impair OMP recognition by SurA, with potential implications for the development of new anti-bacterial therapies that target OM biogenes