Scientific Report 2006

Molecular Biology

Nuclear Magnetic Resonance Studies of the Structure and Dynamics of Enzymes

H.J. Dyson, P.E. Wright, S.H. Bae, D. Boehr, G. Kroon, M. Martinez-Yamout, N.E. Preece, S.C. Sue, L.M. Tuttle, Y. Yao, L.L. Tennant, J. Chung, C.L. Brooks, S.J. Benkovic,* A. Holmgren,** E.A. Komives***

* Pennsylvania State University, University Park, Pennsylvania
** Karolinska Institutet, Stockholm, Sweden
*** University of California, San Diego, California

We use site-specific information on structure and dynamics obtained via nuclear magnetic resonance (NMR) to further the understanding of protein function. We focus on the mechanism of enzymes and the relationship between dynamics and function in a number of medically important systems.

Dynamics in Enzyme Action

Dynamic processes are implicit in the catalytic function of all enzymes. We use state-of-the-art NMR methods to elucidate the dynamic properties of several enzymes. New methods have been developed for analysis of NMR relaxation data for proteins that tumble anisotropically and for analysis of slow timescale motions.

Dihydrofolate reductase plays a central role in folate metabolism and is the target enzyme for a number of antibacterial and anticancer agents. ¹⁵N relaxation experiments on dihydrofolate reductase from Escherichia coli revealed a rich diversity of backbone dynamical features for a broad range of timescales (picoseconds to milliseconds).

A major focus is on the characterization of all intermediates in the dihydrofolate reductase reaction cycle. We have identified functionally important motions in loops that control access to the active site of dihydrofolate reductase on timescales similar to those of the hydride transfer chemistry and the rate-determining step of product release. These motions differ in amplitude and timescale depending on the presence of substrate and/or cofactor in the active site, priming the nicotinamide ring of the cofactor and the pterin ring of the substrate for hydride transfer. In addition, measurements of the population distribution of aliphatic side-chain rotamers provided evidence for coupled motion of active-site side chains that could enhance the catalytic process.

Most recently, we used relaxation dispersion measurements to obtain direct information on microsecond-millisecond timescale motions in dihydrofolate reductase, allowing us to characterize the structures of excited states involved in some of these catalysis-relevant processes. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a timescale that is directly relevant to the structural transitions involved in progression through the catalytic cycle (Fig. 1).

Fig 1. Schematic diagram showing the energy landscape of dihydrofolate reductase catalysis. Ground state (larger) and higher energy (smaller) structures of each intermediate in the cycle, modeled on published x-ray structures are shown. For each intermediate in the catalytic cycle, the higher energy conformations detected in the relaxation dispersion experiments resemble the ‘ground-state’ conformations of adjacent intermediates. Rate constants for the interconversion between the complexes, measured by pre–steady state enzyme kinetics at 298 K, pH6 are indicated with gray arrows, while the rates measured in relaxation dispersion experiments are shown with black arrows. From Boehr et al., Science 313:1638, 2006. Reprinted with permission from AAAS.

Dihydrofolate reductase is also the test system for a series of experiments to address the question, If all of the chemistry goes on at the active site, what is the purpose of the rest of the enzyme? We are using chimeric mutants, synthesized by our collaborator S.J. Benkovic, Pennsylvania State University, by using a library approach. The purpose of these experiments is to test the hypothesis that local variations in amino acid sequence, 3-dimensional structure, and polypeptide chain dynamics strongly influence the local interactions that mediate enzyme catalysis and may constitute the essential circumstance that allows enzymes to achieve high turnover rates as well as exquisite specificity in their reactions. A combination of NMR structure and dynamics measurements, single-molecule fluorescence measurements, and analysis of the catalytic steps in these mutant proteins will provide new insights into the role of the protein in enzyme catalysis.

Structure and Dynamics of Prion Variants

Onset of prion diseases is caused by conversion of the cellular prion protein PrP^C into an abnormally folded isoform, PrP^Sc, that has the same primary structure as PrP^C but a totally different 3-dimensional conformation. The abnormally folded (“scrapie”) form of the protein is associated with several diseases, including scrapie in sheep, bovine spongiform encephalopathy (mad cow disease), and human Creutzfeldt-Jakob disease and other inherited prion diseases. We are gathering information on the mechanism of PrP^Sc formation that can be obtained from structural and dynamic studies of mutant prion proteins corresponding to inherited prion diseases.

Individuals carrying familial mutations such as P102L (P101L in our study) are more susceptible than those without such mutations to prion disease. On the other hand, sheep or humans carrying Q167R and/or Q218K mutations are resistant to scrapie and Creutzfeldt-Jakob disease, respectively. We are using the protease-resistant cores of wild-type and mutant mouse prion proteins to study the structural and dynamic basis of PrP^C-to-PrP^Sc conversion in inherited prion diseases. The core is sufficient to transmit infectivity.

Dynamics and the Function of IκBα

It is becoming increasingly clear that the function of many systems in living cells depends not only on the structures of the components but also on their flexibility. Numerous examples exist in which components of an important biological interaction are unstructured or partly structured. In addition, even those interacting molecules that can be classified as “folded” have areas of mobility. Often, these areas are located precisely in the active site of an enzyme or in the binding site of an interacting molecule.

A central molecular interaction in cellular control is the interaction between the nuclear transcription factor NF-κB and its inhibitor IκBα. IκBα consists of a series of ankyrin repeats, which appear to have differential mobility. Using hydrogen-deuterium exchange and mass spectrometry, our collaborator E.A. Komives, University of California, San Diego, found that the second, third, and fourth ankyrin repeats of IκBαare well folded, whereas the fifth and sixth repeats, apparently with exactly the same structure, are highly dynamic. These observations prompt a number of questions: Are the motions inferred from the hydrogen-deuterium mass spectrometry experiments also reflected in the backbone and side-chain dynamics of the protein, as measured by NMR relaxation? Are the motions still present in the IκBα–NF-κB complex? Are they necessary for complex formation, so that if they are damped out, for example, by site-directed mutagenesis at appropriate positions, is the formation of the complex disfavored? To answer these questions, we are doing a series of NMR experiments on IκBαand its complexes with NF-κB.

Publications

Boehr, D.D., Dyson, H.J., Wright, P.E. An NMR perspective on enzyme dynamics. Chem. Rev. 106:3055, 2006.

Boehr, D.D., McElheny, D., Dyson, H.J., Wright, P.E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638, 2006.