Scientific Report 2005

Molecular Biology

Structure and Function of Proteins as Molecular Machines

E.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau, R.M. Brudler, T. Cross, E.D. Garcin, C. Hitomi, K. Hitomi, L. Holden, C.J. Kassmann, I. Li, M.E. Pique, M.E. Stroupe, J.L. Tubbs, T.I. Wood

Our goals are to understand how proteins function as molecular machines. We use structural, molecular, and computational biology to study proteins of biological and biomedical interest, especially proteins that work synergistically with coupled chromophores, metal ions, or other cofactors.

Photoactive Proteins and Circadian Clocks

To understand in atomic detail how 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 proteins of the photolyase and cryptochrome family catalyze DNA repair or act in circadian clocks. To understand the protein photocycle (Fig. 1), we combined our ultra-high-resolution and time-resolved crystallographic structures of the dark state and 2 photocycle intermediates of PYP with site-directed mutagenesis; ultraviolet-visible spectroscopy; time-resolved Fourier transform infrared spectroscopy; deuterium hydrogen exchange mass spectrometry, in collaboration with V. Woods, University of California, San Diego; and quantum mechanical and electrostatic computational methods, in collaboration with L. Noodleman, Department of Molecular Biology.

Fig. 1. Changes in the flexibility and mobility of PYP during its light cycle revealed by mapping the results of hydrogen-deuterium exchange mass spectrometry analyses (gray-scale shading) onto the x-ray crystallographic structure (ribbon showing overall protein fold). In the signaling state, regions of the protein including the N terminus are released for protein-protein interactions.

Cryptochrome flavoproteins are homologs of light-dependent DNA repair photolyases that function as blue-light receptors in plants and as components of circadian clocks in animals. We determined the first crystallographic structure of a cryptochrome, which revealed commonalities with photolyases in DNA binding and redox-dependent function but showed differences in active-site and interaction surface features. New structures of photolyases from 2 other branches of the photolyase/cryptochrome family that repair cyclobutane pyrimidine dimers and photoproducts helped us decipher the cryptic structure, function, and evolutionary relationships of these fascinating redox-active proteins.

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 of these proteins are not understood. Our crystallographic, dynamical light scattering and small-angle x-ray scattering studies revealed that KaiB self-assembles into a tetramer (Fig. 2).

Fig. 2. The tetrameric assembly of the cyanobacterial circadian clock protein KaiB revealed by small-angle x-ray scattering (experimentally determined shape) and x-ray crystallography (ribbon showing protein fold).

We are also studying clock proteins with PYP-like and 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.

Metalloenzyme Structure and Function

Superoxide dismutases (SODs) act as master regulators of intracellular free radicals and reactive oxygen species by transforming superoxide to oxygen and hydrogen peroxide. Novel nickel SODs assemble into hollow spheres composed of six 4-helix bundle subunits. The 9 N-terminal residues fold into a unique nickel hook motif that shows promise as a detectable metal ion–binding tag in protein purification and structure determination.

Our crystallographic structures of classic copper-zinc SODs from mammals, bacterial symbionts, and pathogens revealed striking differences in the enzyme assembly and in the loops flanking the active-site channel, despite the shared β-barrel subunit fold, catalytic metal center, and electrostatic enhancement of activity. With J. Tainer, Department of Molecular Biology, we determined structures of mutant human SODs found in patients with the disease amyotrophic lateral sclerosis (Lou Gehrig disease), and proposed a hypothesis for how single-site mutations cause this fatal neurodegenerative disease.

To synthesize nitric oxide, a cellular signal and defensive cytotoxin, nitric oxide synthases (NOSs) require calmodulin-orchestrated interactions between their catalytic, heme-containing oxygenase module and their electron-supplying reductase module. Crystallographic structures of wild-type and mutant NOS oxygenase dimers with substrate, intermediate, inhibitors, cofactors, and cofactor analogs, determined in collaboration with D. Stuehr, the Cleveland Clinic, Cleveland, Ohio, and J. Tainer, provided insights into the catalytic mechanism and dimer stability.

Our structure-based drug design projects are aimed at selectively inhibiting inducible NOS, to prevent inflammatory disorders, or neuronal NOS, to prevent migraines, while maintaining blood pressure regulation by endothelial NOS. We integrated biochemical data with our structures of NOS oxygenase, NOS reductase, and calmodulin in complex with peptides derived from NOS to propose a model for the assembled holoenzyme that provides a moving-domain mechanism for electron flow from NADPH through 2 flavin cofactors to the heme. Our structure of the NOS reductase provides new insights into the complex regulatory mechanisms of this enzyme family.

Metalloprotein Design

An ultimate goal for protein engineers is to design and construct new protein variants with desirable catalytic or physical properties. As members of the Scripps Research Metalloprotein Structure and Design Group, we are testing our understanding of the affinity, selectivity, and activity of metal ions by transplanting metal sites from structurally characterized metalloproteins into new protein scaffolds. To aid our design efforts, we have organized quantitative information and interactive viewing of protein metal sites at the Metalloprotein Database and Browser (available at http://metallo.scripps.edu).

For green fluorescent protein and the homologous red fluorescent protein, we designed, constructed, and characterized metal-ion biosensors in which binding of metal ions is signaled by changes in the spectroscopic properties of the naturally occurring fluorophores. The green fluorescent protein scaffold provides advantages over existing probes by allowing optimization with random mutagenesis, noninvasive expression in living cells, and targeting to specific cellular locations. By completing the metalloprotein design cycle from prediction to highly accurate structures, we can rigorously evaluate and improve our algorithms for the design of metal sites. Our related structural studies of green and red fluorescent protein intermediates in chromophore cyclization and oxidation provide a novel mechanism for the spontaneous synthesis of these tripeptide fluorophores within the protein scaffold.

Publications

Barondeau, D.P., Getzoff, E.D. Structural insights into protein-metal ion partnerships. Curr. Opin. Struct. Biol. 14:765, 2004.

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry. Biochemistry 44:1960, 2005.

Dunn, A.R., Belliston-Bittner, W., Winkler, J.R., Getzoff, E.D., Stuehr, D.J., Gray, H.B. Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide synthase. J. Am. Chem. Soc. 127:5169, 2005.

Hitomi, K., Oyama, T., Han, S., Arvai, A.S., Getzoff, E.D. Tetrameric architecture of the circadian clock protein KaiB: a novel interface for intermolecular interactions and its impact on the circadian rhythm. J. Biol. Chem. 280:19127, 2005.

Stroupe, M.E., Getzoff, E.D. The role of siroheme in sulfite and nitrite reductases. In: Tetrapyrroles: Their Birth, Life and Death. Warren, M.J., Smith, A. (Eds.). Landes Bioscience, Georgetown, Tex, in press.

Stuehr, D.J., Wei, C.C., Santolini, J., Wang, Z., Aoyagi, M., Getzoff, E.D. Radical reactions of nitric oxide synthases. In: Free Radicals: Enzymology, Signaling, and Disease. Cooper, C.E., Wilson, M.T., Darley-Usmar, V.H. (Eds.). Portland Press, London, 2004, p. 39. Biochemical Society Symposia, Vol. 71.

Tiso, M., Konas, D.W., Panda, K., Garcin, E.D., Sharma, M., Getzoff, E.D., Stuehr, D.J. C-terminal tail residue ARG1400 enables NADPH to regulate electron transfer in neuronal nitric oxide synthase. J. Biol. Chem., in press.

Tubbs, J.L., Tainer, J.A., Getzoff, E.D. Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation. Biochemistry 44:9833, 2005.

Vevodova, J., Graham, R.M., Raux, E., Schubert, H.L., Roper, D.I., Brindley, A.A., Scott, A.I., Roessner, C.A., Stamford, N.P., Stroupe, M.E., Getzoff, E.D., Warren, M.J., Wilson, K.S. Structure/function studies on an S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis. J. Mol. Biol. 344:419, 2004.

Wei, C.C., Wang, Z.Q., Durra, D., Hemann, C., Hille, R., Garcin, E.D., Getzoff, E.D., Stuehr, D.J. The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation. J. Biol. Chem. 280:8929, 2005.