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Instruments / Technical Area	Publication Details	Published
	Phylogenetic analysis with prediction of cofactor or ligand binding for Pseudomonas aeruginosa PAS and cache domains Andrew Hutchin , Charlotte Cordery , Martin A. Walsh , Jeremy S. Webb , Ivo Tews Microbiology Spectrum 9 DOI: 10.1128/spectrum.01026-21 Open Access Show Abstract ▼ Abstract: PAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture.	Dec 2021
I03-Macromolecular Crystallography	An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity Vincent Debruycker , Andrew Hutchin , Matthieu Masureel , Emel Ficici , Chloé Martens , Pierre Legrand , Richard A. Stein , Hassane S. Mchaourab , José D. Faraldo-Gómez , Han Remaut , Cedric Govaerts Nature Structural & Molecular Biology 10 DOI: 10.1038/s41594-020-0464-y Diamond Proposal Number(s): [12718] Show Abstract ▼ Abstract: Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.	Jul 2020
	Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa Yu-Ming Cai , Andrew Hutchin , Jack Craddock , Martin A. Walsh , Jeremy S. Webb , Ivo Tews Scientific Reports 10 DOI: 10.1038/s41598-020-63008-5 Open Access Show Abstract ▼ Abstract: In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.	Apr 2020
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases Dom Bellini , Sam Horrell , Andrew Hutchin , Curtis Phippen , Richard W. Strange , Yuming Cai , Armin Wagner , Jeremy S. Webb , Ivo Tews , Martin A. Walsh Scientific Reports 7 DOI: 10.1038/srep42166 Diamond Proposal Number(s): [11175, 8663, 8889] Open Access Show Abstract ▼ Abstract: The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.	Feb 2017

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