title: Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.
abstract: How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Amar
authors.initials: A
authors.lastname: Joshi
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Rhys
authors.initials: R
authors.lastname: Grinter
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Inokentijs
authors.initials: I
authors.lastname: Josts
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Justyna A
authors.initials: JA
authors.lastname: Wojdyla
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Edward D
authors.initials: ED
authors.lastname: Lowe
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Renata
authors.initials: R
authors.lastname: Kaminska
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Connor
authors.initials: C
authors.lastname: Sharp
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: McCaughey
authors.affiliation: WestCHEM, School of Chemistry, College of Science and Engineering, University of Glasgow, Glasgow G12 8QQ, UK; Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Aleksander W
authors.initials: AW
authors.lastname: Roszak
authors.affiliation: Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Cogdell
authors.affiliation: School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Olwyn
authors.initials: O
authors.lastname: Byron
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Electronic address: daniel.walker@glasgow.ac.uk.
authors.forename: Daniel
authors.initials: D
authors.lastname: Walker
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address: colin.kleanthous@bioch.ox.ac.uk.
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003087:Colicins / Q000378:metabolism; D018360:Crystallography, X-Ray; D003851:Deoxyribonucleases / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D009154:Mutation; D010802:Phylogeny; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011709:Pyocins / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D003087:Colicins; D046912:Multiprotein Complexes; D011709:Pyocins; C080280:pyocin AP41; D003851:Deoxyribonucleases
keywords: P aeruginosa.; bacteriocin; immunity protein; pyocin AP41; pyocin S2