Interconversion of Functional Motions between Mesophilic and Thermophilic Adenylate Kinases

Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK), for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō) simulation of mesophilic AK from E. coli (AKmeso) to simulations of thermophilic AK from Aquifex aeolicus (AKthermo). In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O) ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS) ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A “heated” simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the “corresponding states” hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways. Author Summary: Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK), which undergoes chemically rate-limiting domain motions coupled to substrate binding. Since mesophiles and thermophiles often differ in functionally important motions, we compare coarse-grained simulations of AKmeso and AKthermo as well as several proline and glycine mutational variants designed to interconvert the dynamics. As might be expected, both domain motions and local unfolding motions are reduced in AKthermo relative to AKmeso. In AKthermo, both of these types of motions can be partially shifted toward more flexible AKmeso by heating or by mutating hinge prolines. However, only mutation to highly flexible glycine produces motions like those of AKmeso. Thus, the rate-limiting global transition likely depends on a combination of hinge flexibility and stability within the LID and NMP domains. Finally, this mutagenic framework can inform the rational design of flexibility and allostery in other proteins toward engineering novel biological control systems.