title: Structural reconstruction of protein ancestry.
abstract: Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia.|Faculty of Medicine, St. Vincent's Clinical School, University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Romain
authors.identifier: 0000-0003-4210-9613
authors.initials: R
authors.lastname: Rouet
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia.|Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: David B
authors.initials: DB
authors.lastname: Langley
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia.|Faculty of Medicine, St. Vincent's Clinical School, University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Schofield
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia.|Faculty of Medicine, St. Vincent's Clinical School, University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Mary
authors.initials: M
authors.lastname: Christie
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia.|Faculty of Medicine, St. Vincent's Clinical School, University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Brendan
authors.initials: B
authors.lastname: Roome
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Porebski
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Ashley M
authors.identifier: 0000-0003-2943-9044
authors.initials: AM
authors.lastname: Buckle
authors.affiliation: Research School of Chemistry, Australian National University, Acton, ACT 2601, Australia.
authors.forename: Ben E
authors.initials: BE
authors.lastname: Clifton
authors.affiliation: Research School of Chemistry, Australian National University, Acton, ACT 2601, Australia.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Jackson
authors.affiliation: Faculty of Medicine, St. Vincent's Clinical School, University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.|Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Daniela
authors.initials: D
authors.lastname: Stock
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia; d.christ@garvan.org.au.|Faculty of Medicine, St. Vincent's Clinical School, University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Christ
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics; D009113:Muramidase / Q000737:chemistry; D010802:Phylogeny; D019056:Receptors, Polymeric Immunoglobulin / Q000737:chemistry* / Q000235:genetics; D014714:Vertebrates / Q000235:genetics / Q000276:immunology
chemical_list: D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D019056:Receptors, Polymeric Immunoglobulin; C421864:hen egg lysozyme; D009113:Muramidase
keywords: antibody; directed evolution; homodimer; protein evolution; protein structure