Modifying a covarying protein-DNA interaction changes substrate preference of a site-specific endonuclease.

Marc Laforet,Kun Zhang,Christopher M Brown,David R Edgell,Murray S Junop,Gregory B Gloor,Michael Vu,Thomas A Mcmurrough

doi:10.1093/nar/gkz866

Abstract

Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein–DNA covariation. Directed evolution experiments of one predicted pair, 227/+3, revealed variants with surprising shifts in I-OnuI substrate preference at the central 4 bases where cleavage occurs. Structural studies showed significant remodeling distant from the covarying position, including restructuring of an inter-hairpin loop, DNA distortions near the scissile phosphates, and new base-specific contacts. Our findings are consistent with a model whereby the functional impacts of covariation can be indirectly propagated to neighboring residues outside of direct contact range, allowing meganucleases to adapt to target site variation and indirectly expand the sequence space accessible for cleavage. We suggest that some engineered meganucleases may have unexpected cleavage profiles that were not rationally incorporated during the design process.

Highlights

Mechanisms that regulate enzyme specificity may not be fully apparent from structural or biochemical studies
Molecular covariation analysis utilizes multiple sequence alignments to predict intramolecular amino acid codependencies with the hypothesis that covarying residues contribute to protein structure and function [1,2,3]
To identify putative specificity-determining features of meganucleases that are distinct from those previously identified by structural studies, we used computational MI methods to predict protein–DNA covariation from multiple sequence alignments

Summary

Introduction

Mechanisms that regulate enzyme specificity may not be fully apparent from structural or biochemical studies. Molecular covariation analysis utilizes multiple sequence alignments to predict intramolecular amino acid codependencies with the hypothesis that covarying residues contribute to protein structure and function [1,2,3]. Predicted residue pairs often lie within contact distance of each other providing a straightforward interpretation of covariation where direct contacts disrupted by substitution of one residue can be functionally compensated for by covariation in the other residue [4]. When applied to protein families with rapidly evolving DNA-binding interfaces [9,10], covariation predictions of intermolecular protein–DNA dependencies could reveal direct and indirect mechanisms that regulate DNA interactions to provide useful starting points for engineering new specificity

Methods

Results

Conclusion