J.T. Biel

Keep on moving: Discovering and perturbing the conformational dynamics of enzymes

Conspectus Because 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.

Flexibility and Design: Conformational Heterogeneity along the Evolutionary Trajectory of a Redesigned Ubiquitin

Although protein design has been used to introduce new functions, designed variants generally only function as well as natural proteins after rounds of laboratory evolution. One possibility for this pattern is that designed mutants frequently sample nonfunctional conformations. To test this idea, we exploited advances in multiconformer modeling of room-temperature X-ray data collection on redesigned ubiquitin variants selected for increasing binding affinity to the deubiquitinase USP7. Initial core mutations disrupt natural packing and lead to increased flexibility. Additional, experimentally selected mutations quenched conformational heterogeneity through new stabilizing interactions. Stabilizing interactions, such as cation-pi stacking and ordered waters, which are not included in standard protein design energy functions, can create specific interactions that have long-range effects on flexibility across the protein. Our results suggest that increasing flexibility may be a useful strategy to escape local minima during initial directed evolution and protein design steps when creating new functions.

New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering

Allostery is an inherent feature of proteins and provides alternative routes to regulating function. Small-molecule allosteric inhibitors are often desirable; however, it remains challenging to identify surface sites in proteins which can bind small molecules and modulate function. We identified new allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal “hidden” low-occupancy conformational states for protein and ligands. Our results converge on a new allosteric site that is conformationally coupled to the active-site WPD loop, a hotspot for fragment binding, not conserved in the closest homolog, and distinct from other recently reported allosteric sites in PTP1B. Targeting this site with covalently tethered molecules allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure revealed by multitemperature crystallography can be exploited for developing allosteric modulators.