Scientific Report 2005

Molecular Biology

Nuclear Magnetic Resonance Studies of the Structure and Dynamics of Enzymes

H.J. Dyson, P.E. Wright, D. Boehr, M.O. Ebert, G. Kroon, J. Lansing, C.W. Lee, M. Martinez-Yamout, D. McElheny, N.E. Preece, K. Sugase, H.S. Won, Y. Yao, L.L. Tennant, J. Chung, C.L. Brooks, S.J. Benkovic,* A. Holmgren**

* Pennsylvania State University, University Park, Pennsylvania
** Karolinska Institutet, Stockholm, Sweden

We use site-specific information from nuclear magnetic resonance (NMR) to further the understanding of enzyme function through study of enzyme structure and dynamics. We focus on the mechanisms of enzymes and the relationship between dynamics and function in cellular control by thiol-disulfide chemistry.

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 time scale motions.

Dihydrofolate reductase plays a central role in folate metabolism and is the target enzyme for a number of anticancer agents. ¹⁵N relaxation experiments on dihydrofolate reductase from Escherichia coli revealed a rich diversity of backbone dynamics for a broad range of time scales (picoseconds to milliseconds). These studies were extended to additional intermediates in the reaction cycle and to forms of the enzyme with mutations at various motional “hot spots.”

In addition, we are using ²H relaxation measurements in triple-labeled dihydrofolate reductase to elucidate the dynamics of critical active-site side chains. So far, we have identified functionally important motions in loops that control access to the active site of the reductase on the same time scale as the hydride transfer chemistry. These motions become attenuated once the NADPH cofactor is bound in the active site, locking the nicotinamide ring in a geometry conducive to hydride transfer to substrate. We also found evidence of motion of active-site side chains that are implicated in the catalytic process.

Most recently, we used relaxation dispersion measurements to obtain direct information on microsecond-millisecond time scale motions in dihydrofolate reductase, allowing us to characterize the structures of the 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 time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.

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 will use a series of 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.

Redox Control by Thiol-Disulfide Chemistry

Many cellular functions are regulated by thiol-disulfide chemistry. The importance of redox chemistry, particularly disulfide-dithiol equilibria, in cellular control mechanisms has only recently been recognized. For example, the chaperone heat-shock protein 33 (Hsp33) is regulated by a redox switch; the C-terminal domain of Hsp33 contains cysteines that are reduced and bound to zinc under normoxic conditions, but upon oxidation, the zinc is lost and disulfide bonds form. Interestingly, the zinc-bound form of the C-terminal domain is well structured, with a distinctive fold. NMR studies revealed that upon oxidation, the C-terminal domain becomes unstructured. We think that this loss of local structure exposes a dimerization site. Thus, under oxidative stress conditions, the chaperone dimerizes to the active form.

We did an extensive study of the structural basis for the activity of several thiol-disulfide enzymes. Thioredoxin, a small, 108-residue thiol-disulfide oxidoreductase, has many functions in the cell, including reduction of ribonucleotides to form deoxyribonucleotides for DNA synthesis. A primary function of thioredoxin in the cell is as a protein disulfide reductase, a function vital for the prevention of misfolded proteins in vivo. The E coli thioredoxin system has been fully characterized by using NMR, including the calculation of high-resolution structures for both the oxidized (disulfide) and the reduced (dithiol) forms of the protein.

Using backbone dynamics and amide proton hydrogen exchange, we found that functional differences in phage systems between oxidized and reduced thioredoxin were due to differences in the flexibility of the molecules rather than to structural differences. We also delineated the mechanism of E coli thioredoxin. We found that the reduction reaction of thioredoxin depends critically on the movement of protons, during the 2-electron–2-proton transfer reaction, as a substrate disulfide is reduced. We are investigating a variant E coli thioredoxin with an N-terminal extension that binds zinc. This exciting new molecule may be another example of a redox-active, zinc-binding protein, previously exemplified by the redox-switch domain of the chaperone Hsp33.

Glutaredoxins are another major class of thiol-disulfide regulatory proteins. We recently determined the structure of glutaredoxin-2 from E coli. This protein appears to be a link between the glutaredoxin-thioredoxin class of small thiol-active proteins and the extensive glutathione-S-transferase class of detoxification enzymes. Glutaredoxins are thought to be involved in the processes that result in the attachment and removal of glutathione and nitrosyl groups from redox-active proteins. These processes, together with the formation of disulfide bonds, regulate the activity of redox-active proteins such as the transcription factor OxyR, which we also study.

Publications

Chen, J., Won, H.-S., Im, W., Dyson, H.J., Brooks, C.L. III. Generation of native-like protein structures from limited NMR data, modern force fields and advanced conformational sampling. J. Biomol. NMR 31:59, 2005.

McElheny, D., Schnell, J.R., Lansing, J.C., Dyson, H.J., Wright, P.E. Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc. Natl. Acad. Sci. U. S. A. 102:5032, 2005.

Venkitakrishnan, R.P., Zaborowski, E., McElheny, D., Benkovic, S.J., Dyson, H.J., Wright, P.E. Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle. Biochemistry 43:16046, 2004.