Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Abstract

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

“…The data for FGFR1 KD (Figs 1b and 5a–c) are in sharp contrast with recent observations obtained for a metabolic phosphoglycerate kinase (PGK)40. MD simulations and smFRET show equilibrium between two populations (“expanded” and “compact”) for ligand-free PGK; substrate binding only slightly shifts the equilibrium toward the compact state.…”

“…Furthermore, we have also addressed a major obstacle of non-disruptive labeling of target regions that are key for the activation process, and therefore functionally sensitive, by applying the genetically encoded, site-specific incorporation of unnatural amino acids (UAAs), coupled with rapid and highly specific labeling with a chemical reporter33343536373839. Importantly, using this novel type of FRET-based construct, combined with MD simulations, we show that FGFR1 KD displays switch-like activation owing to a high free energy barrier between its non-phosphorylated and phosphorylated states, contrasting some recent examples for other kinases supporting preexisting equilibrium242640.…”

“…MD simulations and smFRET show equilibrium between two populations (“expanded” and “compact”) for ligand-free PGK; substrate binding only slightly shifts the equilibrium toward the compact state. These data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations and that catalytically relevant conformations are, to a large extent, already occurring in ligand-free PGK40. MD simulations have also been performed for several signaling protein kinases including EGFR, Abl, Src and B-Raf222324252628.…”

Homodimerization enhances both sensitivity and dynamic range of the ligand‐binding domain of type 1 metabotropic glutamate receptor

“…This entropic model is consistent with the recent observation of a highly dynamic population of the intermediate closed/open state in the LBD of mGluR3 in solution . Data from structurally related model proteins, such as maltose‐binding protein and phosphoglycerate kinase have also revealed large‐amplitude oscillations around the hinge axis in the unliganded state whose amplitude is reduced upon either ligand binding or mutations that rigidify the hinge. However, it is also possible that the closed/open dimer in mGluR1 LBD is favored over the open–open one enthalpically.…”

Adaptability of protein structures to enable functional interactions and evolutionary implications

“…This highly versatile protein adopts a variety of conformations while binding its substrates, and these are simply those sampled along one or two principal directions of motions accessible to the unbound ubiquitin, also seen by NMR residual dipolar coupling. A more recent example is the single-molecule Förster resonance energy transfer analysis of phosphoglycerate kinase (PGK) dynamics by Fitter and coworkers [23••]. In that study, Fitter and coworkers elegantly showed that (i) the experimentally detected functional (hinge-bending) motions of the enzyme are encoded by the fold, as predicted by ENMs, and (ii) those motions are already performed in the ligand-free state of PGK domains, prior to substrate-binding.…”