Biochemistry Publications
A Unified Genetic, Computational and Experimental Framework Identifies Functionally Relevant Residues of the Homing Endonuclease I-BmoI
Document Type
Article
Publication Date
4-2010
Journal
Nucleic Acids Research
Volume
38
Issue
7
First Page
2411
Last Page
2427
URL with Digital Object Identifier
http://dx.doi.org/10.1093/nar/gkp1223
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.