A Master Switch Couples Mg2+-Assisted Catalysis to Domain Motion in B. Stearothermophilus Tryptophanyl-tRNA Synthetase

Abstract

Tryptophanyl-tRNA synthetase (TrpRS) uses conformation-dependent Mg2+ activation to couple catalysis of tryptophan activation to specific, functional domain movements. Rate acceleration by Mg2+ requires ∼ −6.0 kcal/mole in protein⋅Mg2+ interaction energy, none of which arises from the active site. A highly cooperative interaction between Mg2+ and four residues from a remote, broadly conserved motif that mediates the shear of domain movement: (i) destabilizes the pre-transition state conformation, thereby (ii) inducing the Mg2+ to stabilize the transition state for kcat by ∼ −5.0 kcal/mole. Thermofluor studies of differential conformational stability in single mutants to four residues in the motif verify that the conformational changes impact catalysis. Steady-state kinetic analysis of all possible combinations of these four mutations assayed with Mg2+ and Mn2+ comprises a 5-dimensional multi-mutant thermodynamic cycle. The coupling energies of this cycle are dominated by the 5-way interaction between all four residues of the remote motif and the metal. The coupling energy of this high-order interaction provides 5 kcal/mole in transition state stabilization. This value represents the equilibrium constant for the five components. The thermodynamic differential stability analysis implies that the relevant process is the coupling between the conformational change and the metal activation. Cooperative, long-range conformational effects on the metal therefore convert an inactive Mg2+ coordination into one that can stabilize the transition state if, and only if, domain motion occurs. Conformation-dependent Mg2+ activation, analogous to the escapement in mechanical clocks, explains directional coupling. We propose that similar metal-dependent conformational coupling occurs in other transducing NTPases that also couple catalysis of NTP utilization to functionally relevant conformational changes.