title: A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI.
abstract: Insight into protein structure and function is best obtained through a synthesis of experimental, structural and bioinformatic data. Here, we outline a framework that we call MUSE (mutual information, unigenic evolution and structure-guided elucidation), which facilitated the identification of previously unknown residues that are relevant for function of the GIY-YIG homing endonuclease I-BmoI. Our approach synthesizes three types of data: mutual information analyses that identify co-evolving residues within the GIY-YIG catalytic domain; a unigenic evolution strategy that identifies hyper- and hypo-mutable residues of I-BmoI; and interpretation of the unigenic and co-evolution data using a homology model. In particular, we identify novel positions within the GIY-YIG domain as functionally important. Proof-of-principle experiments implicate the non-conserved I71 as functionally relevant, with an I71N mutant accumulating a nicked cleavage intermediate. Moreover, many additional positions within the catalytic, linker and C-terminal domains of I-BmoI were implicated as important for function. Our results represent a platform on which to pursue future studies of I-BmoI and other GIY-YIG-containing proteins, and demonstrate that MUSE can successfully identify novel functionally critical residues that would be ignored in a traditional structure-function analysis within an extensively studied small domain of approximately 90 amino acids.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Kleinstiver
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Fernandes
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Gloor
authors.forename: David R
authors.initials: DR
authors.lastname: Edgell
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D020134:Catalytic Domain; D019295:Computational Biology / Q000379:methods*; D053837:DNA Cleavage; D003627:Data Interpretation, Statistical; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D000596:Amino Acids; D004706:Endodeoxyribonucleases; C432135:I-BmoI endonuclease