Computational and experimental investigation of allosteric communication in the transcriptional regulator NikR

Abstract

The Ni²⁺ and DNA binding protein NikR is involved in nickel regulation in Escherichia coli through transcriptional repression of the NikABCDE nickel permease. NikR is a homotetramer and each chain contains both a DNA binding ribbon-helix-helix (RHH) domain and a Ni ²⁺ binding regulatory ACT (aspartokinase, chorismate mutase, TyrA) fold. Work herein combines computational modeling of NikR structure with experimental studies aimed at understanding allosteric communication between the ACT and RHH domains. Hydrogen/deuterium exchange mass spectrometry shows a Ni ²⁺ specific NikR conformational change relative to bound Cu ²⁺, Co²⁺, and Zn²⁺. Concurrent coordination geometry and in vivo repressor function studies show that NikR activation is specific to binding Ni²⁺ in square-planar geometry. These results suggest that regions of the NikR structure distal to the Ni²⁺ binding sites are involved in allosteric communication. To help determine important residue interactions within and between the RHH and ACT domains that are involved in allostery, an equilibrium molecular dynamics (MD) simulation is utilized to explore the conformational dynamics of the NikR tetramer. This study includes advances in methods development focused on identifying signatures of allosteric communication in MD simulations. Using two different correlation measures based on fluctuations in atomic position and non-covalent bonding, we identify a potential allosteric communication pathway between the Ni²⁺ and DNA binding sites. We also apply a graph theoretic approach to map the most probable networks of non-covalent contacts connecting the two functionally important binding sites. Several of the residues identified by our analyses have been shown experimentally to be important for NikR function. An additional subset of the selected residues structurally connects experimentally important residues and may help coordinate allosteric communication between the ACT and RHH domains. Based on these analyses and additional structural interpretations, site-directed mutagenesis of E. coli NikR and subsequent characterization of changes in Ni ²⁺ binding and in vivo repressor function of mutants aid our understanding of the role of these residues in allosteric regulation. The combination of computational and experimental methods that are developed or adapted in this study provides a framework for further characterization of NikR, other ACT domain containing proteins, and other allosteric proteins.