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Scientific Report 2008


Molecular Biology




Protein Structures, Activities, and Regulation: The Functioning of Molecular Machines


E.D. Getzoff, A.S. Arvai, E.D. Garcin, C. Hitomi, K. Hitomi, M.D. Kroeger, M.E. Pique, A.J. Pratt, D.S. Shin

To understand how proteins function as molecular machines, we use structural molecular biology to characterize and control proteins. We apply the tools of structural, molecular, and computational biology to analyze proteins of biological and biomedical interest, especially proteins that work synergistically with coupled chromophores, metal ions, or other cofactors.

Nitric Oxide Synthase and a New Approach to Design of Selective Inhibitors

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilation and neurotransmission at low levels and a defensive cytotoxin at higher levels. Synthesis of nitric oxide by NOS requires calmodulin-coordinated interactions between the catalytic, oxygenase, and electron-supplying reductase modules of the enzyme. NOS uses heme, zinc, tetrahydrobiopterin, calcium, NADPH, FMN, and FAD cofactors.

The goals of our structure-based drug design projects are to selectively inhibit inducible NOS, to prevent inflammatory disorders, or neuronal NOS, to prevent migraines, while maintaining blood pressure regulation by endothelial NOS. Our x-ray crystallographic structures of wild-type and mutant NOS oxygenase dimers with substrate, intermediate, inhibitors, cofactors, and cofactor analogs, determined in collaboration with J.A. Tainer, Department of Molecular Biology, and D. Stuehr, the Cleveland Clinic, Cleveland, Ohio, provide insights into both catalytic mechanism and inhibitor selectivity. The nearly complete sequence and structural conservation in the active sites of the 3 NOS isozymes is a significant challenge in the design of isozyme-specific inhibitors. Nevertheless, our latest results indicate that plasticity of distant isozyme-specific residues modulates conformational changes of invariant residues in the substrate-binding site. We applied these results to develop the anchored plasticity approach for the structure-based design of selective inhibitors (Fig. 1). In this approach, conserved binding sites are used to anchor the core of an inhibitor, while distant sequence differences are exploited to provide selectivity.


Fig. 1. Diagram illustrating the anchored-plasticity approach to selective drug design.

Our structure of the neuronal NOS reductase reveals new insights into the complex regulatory mechanisms of this enzyme family. We integrated biochemical data with our structures of NOS oxygenase, NOS reductase, and calmodulin in complex with NOS peptides to propose assembly and mechanistic hypotheses for the holoenzyme. We obtained promising results in support of these hypotheses by using solution small-angle x-ray scattering, which can provide molecular envelopes for macromolecules and macromolecular complexes in solution.

We have proposed a moving-domain mechanism for controlling the rate-limiting flow of electrons from the flavin cofactors of NOS reductase to the catalytic NOS oxygenase heme. Our assembly and mechanistic hypotheses also explain the kinetics of regulatory site-specific phosphorylation and dephosphorylation events that both activate and inactivate synthesis of nitric oxide in vivo. In ongoing research, we are using complementary biochemical, biophysical, and computational methods to define, describe, and understand how NOS chemistry, structure, assembly, dynamics, and protein-protein interactions regulate production of nitric oxide in vivo.

Photoactive Proteins and Circadian Clocks

To understand in atomic detail how chromophore-bound proteins translate sunlight into defined conformational changes for biological functions, we are exploring the reaction mechanisms of the blue-light receptors photoactive yellow protein (PYP), photolyase, and cryptochrome. PYP is the prototype for the Per-Arnt-Sim domain proteins of circadian clocks, whereas FAD-containing proteins of the photolyase/cryptochrome family catalyze DNA repair or act in circadian clocks. To understand the photocycle of PYP and to propose a common mechanism for signaling by Per-Arnt-Sim domains, we combined ultra-high-resolution and time-resolved crystallographic structures of the PYP dark state and 2 photocycle intermediates with site-directed mutagenesis; spectroscopy; deuterium-hydrogen exchange mass spectrometry, in collaboration with V. Woods, University of California, San Diego; and quantum mechanical and electrostatic computational methods, in combination with L. Noodleman, Department of Molecular Biology.

Cryptochrome flavoproteins function as blue-light receptors in plants and as components of circadian clocks in animals. We determined the first crystallographic structure of a cryptochrome; the structure revealed commonalities with the homologous photolyases in DNA binding and redox-dependent function but showed differences in active-site and interaction-surface features. We found that this cryptochrome binds he same antenna cofactor found in a photolyase homolog but uses different amino acid residues to form the cofactor-binding site. Our new structures and spectroscopy, obtained in collaboration with S. Weber, Universität Freiburg in Germany, of cryptochromes and of photolyases from 2 other branches of the photolyase/cryptochrome family that repair cyclobutane pyrimidine dimers and (6-4) photoproducts help us decipher the cryptic structure, function, and evolutionary relationships of these fascinating redox-active proteins. Furthermore, the (6-4) photolyase enzyme provides an excellent model for human cryptochrome.

A simple, but functional, circadian clock can be reconstituted in vitro from the 3 cyanobacterial proteins KaiA, KaiB, and KaiC alone. Yet, the structure and dynamics of the functional assembly are not understood. Our crystallographic, dynamical light scattering and small-angle x-ray scattering studies revealed that KaiB self-assembles into a tetramer. We also study clock proteins with PYP-like Per-Arnt-Sim domains that bind to mammalian cryptochromes. Our goal is to determine the detailed chemistry and atomic structure of these proteins, define their mechanisms of action and interaction, and use our results to understand and regulate biological function.

Superoxide Dismutase

The superoxide radical is a central player in the biology of reactive oxygen and nitrogen intermediates, which mediate signaling and oxidative damage, a key factor in aging and cancer, in vivo. Mutations in human copper zinc superoxide dismutase (SOD), the enzyme that converts superoxide to molecular oxygen and hydrogen peroxide, cause the fatal neurodegenerative disease familial amyotrophic lateral sclerosis, or Lou Gehrig disease. We are analyzing the structural chemistry of SOD to help bridge the gap from protein structures to enzyme stability and activities in vivo. We designed a zinc-free variant of human SOD to help test the role of zinc binding and loss in disease. Our results, obtained in collaboration with J.A. Tainer, support the importance of the stable SOD core structure in preventing amyloid formation and toxic effects. For comparison, we determined the structure and stability of the SOD from the most extreme eukaryotic thermophile known: the deep-sea hydrothermal vent worm Alvinella pompejana.

Publications

Garcin, E.D., Arvai, A.S., Rosenfeld, R.J., Kroeger, M.D., Crane, B.R., Andersson, G., Andrews, G., Hamley, P.J., Mallinder, P.R., Nicholls, D.J., St-Gallay, S.A., Tinker, A.C., Gensmantel, N.P., Mete, A., Cheshire, D.R., Connolly, S., Stuehr, D.J., Åberg, A., Wallace, A.V., Tainer, J.A., Getzoff, E.D. Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nat. Chem. Biol., in press.

Roberts, B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., Anderson, S.R., Bomben, V.C., Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS. J. Mol. Biol. 373:877, 2007.

Yamamoto, J., Tanaka, Y., Hitomi, K., Getzoff, E.D., Iwai, S. Spectroscopic studies on a novel intramolecular hydrogen bond within the (6-4) photoproduct. Nucleic Acids Symp. Ser. (Oxf). Issue 51:79, 2007.

 

Elizabeth D. Getzoff, Ph.D.
Professor



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