Keep on Moving: Discovering and Perturbing the Conformational Dynamics of Enzymes

Abstract

ConspectusBecause living organisms are in constant motion, the word “dynamics” can hold many meanings to biologists. Here we focus specifically on the conformational changes that occur in proteins and how studying these protein dynamics may provide insights into enzymatic catalysis.Advances in integrating techniques such as X-ray crystallography, nuclear magnetic resonance, and electron cryomicroscopy (cryo EM) allow us to model the dominant structures and exchange rates for many proteins and protein complexes. For proteins amenable to atomic resolution techniques, the major questions shift from simply describing the motions to discovering their role in function. Concurrently, there is an increasing need for using perturbations to test predictive models of dynamics–function relationships. Examples are the catalytic cycles of dihydrofolate reductase (DHFR) and cyclophilin A (CypA).In DHFR, mutations that alter the ability of the active site to sample productive higher energy states on the millisecond time scale reduce the rate of hydride transfer significantly. Recently identified rescue mutations restore function, but the mechanism by which they do so remains unclear. The exact role of any changes in the dynamics remains an open question. For CypA, a network of side chains that exchange between two conformations is critical for catalysis. Mutations that lock the network in one state also reduce catalytic activity.A further understanding of enzyme dynamics of well-studied enzymes such as dihydrofolate reductase and cyclophilin A will lead to improvement in ability to modulate the functions of computationally designed enzymes and large macromolecular machines. In designed enzymes, directed evolution experiments increase catalytic rates. Detailed X-ray studies suggest that these mutations likely limit the conformational space explored by residues in the active site. For proteins where atomic resolution information is currently inaccessible, other techniques such as cryo-EM and high-resolution single molecule microscopy continue to advance. Understanding the conformational dynamics of larger systems such as protein machines will likely become more accessible and provide new opportunities to rationally modulate protein function.

Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies

“…The results are consistent with a catalytic mechanism sufficiently described by conformational and electrostatic fluctuations on slower time scales. 36,45,46 The ecDHFR protein creates a geometric and electrostatic catalytic cage where the reaction coordinate is formed by favorable electrostatic apposition of the reactants.…”

Conformational Sub-states and Populations in Enzyme Catalysis

“…The catalytic cycle of Escherichia coli DHFR (EcDHFR) consists of at least five sub-states corresponding to intermediates associated with the substrate (DHF) and cofactor (NADPH) binding, the chemical step of hydride transfer and the product (THF) and spent cofactor (NADP + ) release. Using 15 N spin-relaxation NMR techniques (mostly the rcCPMG described above), Wright and co-workers discovered that at each intermediate along the catalytic cycle represents sampling of populations from multiple conformational sub-states (Figure 7) (Bhabha, Biel, & Fraser, 2015; Boehr, Dyson, et al, 2006; Boehr, McElheny, et al, 2006). …”

The role of protein dynamics in allosteric effects—introduction

“…They showed that free energies of a few kcal/mol can be derived from only a slight stiffening of a few of the many global dynamic modes of motion available to a protein. With the increasing understanding of the importance of dynamics in enzyme catalysis (see the recent virtual special issue of Accounts of Chemical Research on 'Protein Motions in Catalysis'; Bhabha et al 2015;Callender and Dyer 2015;Hanoian et al 2015;Klinman 2015;Kohen 2015;Palmer 2015) and in protein function in general, the importance of dynamics as a contributory mechanism in allosteric effects is now recognised (Jardetzky 1996;Kern and Zuiderweg 2003;Tsai et al 2008).…”