Activation Mechanism of a Signaling Protein at Atomic Resolution from Advanced Computations

Abstract

Advanced computational techniques including transition path sampling and free energy calculations are combined synergistically to reveal the activation mechanism at unprecedented resolution for a small signaling protein, chemotaxis protein Y. In the conventional "Y-T coupling" model for response regulators, phosphorylation induces the displacement of the conserved Thr87 residue through hydrogen-bond formation, which in turn makes it sterically possible for Tyr106 to isomerize from a solvent exposed configuration to a buried rotameric state. More than 160 unbiased activation trajectories show, however, that the rotation of Tyr106 does not rely on the displacement of Thr87 per se. Free energy calculations reveal that the Tyr106 rotation is a low-barrier process in the absence of the Thr87-phosphate hydrogen bond although the rotation is stabilized by the formation of this interaction. The simulations also find that structural change in the β4 − α4 loop does not gate the Tyr106 rotation as suggested previously; rather, the rotation of Tyr106 stabilizes the activated configuration of this loop. The computational strategy used and mechanistic insights obtained have impact on the study of signaling proteins and allosteric systems in general.

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

“…Transition path sampling (TPS) has transformed the computational study of rare events in molecular systems over the last decade (11,12), but its application to complex, biological systems has been limited (24,25,(27)(28)(29). Efficient means for generating initial paths (14) together with informatic methods (13) that we introduced now enable us to harvest a statistically significant number of trajectories of a biomedically important stochastic process in its entirety and identify the features that characterize the ensemble of transition states.…”

“…This number of trajectories is statistically significant given that paths were found to decorrelate within approximately three shooting moves, as estimated by a procedure adapted from ref. 24 [supporting information (SI) Fig. 5].…”

Allostery and cooperativity revisited

“…To gain further mechanistic insights into the activation of CheY, as an example of monomeric protein allostery, molecular dynamics and free energy simulations were used to explore the coupling between various conformational transitions (e.g., the b4Àa4 loop transition, Tyr 106 rotation, and Thr 87 displacement) and phosphorylation in both the wild-type CheY and the T87A mutant (Formaneck et al 2006;Ma and Cui 2007). Using the transition path sampling technique (Bolhuis et al 2002), it has been shown ) that the isomerization of Tyr 106 does not require the displacement of Thr 87 and that the hydrogen bond between Thr 87 and Asp 57 phosphate, an essential element of the ''Y-T'' scheme, is not formed.…”

Evidence Against the “Y–T Coupling” Mechanism of Activation in the Response Regulator NtrC

“…23 Pandini et al also found support for a pre-organized network of allosteric connections between these residues and the functional site of phosphorylation. 24 Alternatively, transition path sampling in combination with potential mean force calculations found that the energy barrier of tyrosine/phenylalanine rotamer exchange was lower than the barrier involved in the inactive/active transition, making the rotameric state of the conserved aromatic residue kinetically independent from the functional conformational transition, and leading them to theorize a role for the tyrosine in the thermodynamic stabilization of the active substate 25; 26 .…”