Arginine Kinase: Joint Crystallographic and NMR RDC Analyses Link Substrate-Associated Motions to Intrinsic Flexibility

Abstract

The phosphagen kinase family, including creatine and arginine kinases, catalyze the reversible transfer of a "high energy" phosphate between ATP and a phospho-guanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of arginine kinase structures was interpreted as a plastic deformation. Here, the structure of Limulus substrate-free arginine kinase is refined against high resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa), and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.2 Å transition state analog complex shows large substrate-induced domain motions which can be broken down into movements of smaller quasi-rigid bodies. The solution state structure of substrate-free arginine kinase is most consistent with an equilibrium of substrate-free and -bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, "substrate-induced" motions are along modes that are intrinsically flexible in the substrate-free enzyme, and likely involve some degree of conformational selection.

The Structure of Lombricine Kinase

“…These motions, measured in the absence of substrates, occur at turnover-commensurate rates (22) at sites that are also implicated in the substrate-associated conformation changes (36). These observations suggest that substrate-associated changes take advantage of modes of flexibility intrinsic to the enzyme and that some of the intrinsic motions may be rate-limiting on turnover.…”

“…The anatomy of conformational change was examined with reference to conformational differences between the transition state analog and substrate-free forms of LpAK, the phosphagen kinase that has the largest of otherwise similar conformational changes through the family (36). The conformational changes in LpAK are approximated well by rigidgroup motions of five subdomains, sometimes referred to as "dynamic domains" (57) because they consist of spatial clusters of residues that need not be contiguous in sequence but that share a common displacement.…”

“…The conformational changes on adoption of the transition state have been examined crystallographically in detail for LpAK and compared with the intrinsic dynamics in the substrate-free state using solution NMR (36). Backbone motions are well approximated by the rigid-group motions of five subdomains.…”

“…Thus, locally the substrate-free loop structure is not conserved and/or is influenced by the crystal environment. Noting that the loop restructuring involves motions (up to 18 Å) larger than typical for the fast (ns-ps) intrinsic dynamics as characterized by NMR in LpAK (36), it is likely that many of the reported loop structures are off the reaction pathway.…”

Protein dynamics and function from solution state NMR spectroscopy

“…There are multiple ways of using RDCs to study the dynamics of proteins. They are well-suited for comparing the structural state of a protein in solution to structures obtained from x-ray crystallography (Bernado & Blackledge, 2004;Bouvignies et al 2005Bouvignies et al , 2006Clore & Schwieters, 2004b;Salvatella et al 2008), as shown elegantly for various states of the HIV-1 protease (Roche et al 2015) and arginine kinase (Niu et al 2011). As an extension of this approach, it is also possible to determine the distribution of structural states in proteins by using ensemble methods (Esteban-Martin et al 2010, 2014; Fenwick et al…”