A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI.
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.
Kleinstiver
Benjamin P
BP
Department of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
Fernandes
Andrew D
AD
Gloor
Gregory B
GB
Edgell
David R
DR
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
D000596:Amino Acids; D004706:Endodeoxyribonucleases; C432135:I-BmoI endonuclease