Scientific Report 2006

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.H. Cross, E.D. Garcin-Hosfield, C. Hitomi, K. Hitomi, C.J. Kassmann, 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.

Nitric Oxide Synthases

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 J. Tainer, Department of Molecular Biology, and D. Stuehr, the Cleveland Clinic, Cleveland, Ohio, 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. Our structure of the neuronal NOS reductase has provided news insights into the complex regulatory mechanisms of this enzyme family.

We have designed and assayed site-directed mutant enzymes that support our mechanistic hypotheses for isozyme-specific inhibition and regulation (Fig. 1). 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 NOS reductase to the NOS oxygenase heme. Preliminary small-angle x-ray scattering measurements in solution provide molecular envelopes for NOS proteins that support our model. Our model also explains how regulatory site-specific phosphorylation and dephosphorylation activate and inactivate nitric oxide synthesis in vivo.

Fig. 1. The NADPH-binding site in the crystallographic structure of the neuronal NOS reductase module. A triad of amino acid residues conserved in NOS reductases and homologous flavoproteins (Tyr1322, Ser1313, and Arg1314) stabilize the 2′ phosphate group (2′P) that distinguishes NADPH from NADH. In contrast, Arg1400 is specific to the calcium-regulated neuronal and endothelial NOS enzymes, in which it performs a isozyme-specific regulatory function. In the absence of calcium-bound calmodulin, Arg1400 helps stabilize the regulatory C-terminal tail, inhibiting nonproductive electron transfer.

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, we combined 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.

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. Recently, we showed that this cryptochrome binds the same antenna cofactor found in a photolyase homolog but uses different residues for the cofactor-binding site. New structures of photolyases from 2 other branches of the photolyase/cryptochrome family that repair cyclobutane pyrimidine dimers and photoproducts help us decipher the cryptic structure, function, and evolutionary relationships of these fascinating redox-active proteins.

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 their biological function and regulation.

Protein Design and Postranslational Modification Chemistry

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 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 (GFP) and the homologous red fluorescent protein (RFP), we designed, constructed, and characterized metal-ion biosensors, in which binding of metal ions is signaled by changes in spectroscopic properties of the naturally occurring fluorophores. Use of GFP allows 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 algorithms for the design of metal sites.

In related research, we discovered that the architecture of GFP and RFP promotes a remarkable range of posttranslational modification chemistry. High-resolution crystallographic structures of GFP and RFP intermediates in fluorophore cyclization and oxidation lead to a novel mechanism for the spontaneous synthesis of this tripeptide fluorophore within the protein scaffold. Remarkably, the same protein architectural features that favor peptide cyclization can drive peptide hydrolysis (Fig. 2) and red shift the spectral properties of the chromophore. Decarboxylation reactions in designed variants of GFP (Fig. 2) support a role for the GFP environment in facilitating formation of radicals and 1-electron chemistry. Together, our results provide the groundwork for the design of proteins with novel catalytic or reporter properties.

Fig. 2. Spontaneous peptide hydrolysis and decarboxylation reactions promoted by the protein architecture of GFP. A, Crystallographic structure of a designed GFP variant reveals peptide-bond cleavage and decarboxylation chemistry at the site of GFP fluorophore synthesis. S65G and Y66S mutations converted the fluorophore tripeptide SYG sequence to GSG. The simulated annealing omit electron density map (mesh) clearly shows the resultant break in the polypeptide chain at this site. B, Corresponding reaction and posttranslational products for this self-cleaving GFP variant.

Publications

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone fragmentation, hydrolysis, and decarboxylation. J. Am. Chem. Soc. 128:4685, 2006.

Barondeau, D.P., Tainer, J.A., Getzoff, E.D. Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis. J. Am. Chem. Soc. 128:3166, 2006.

Brudler, R., Gessner, C.R., Li, S., Tyndall, S., Getzoff, E.D., Woods, V.L., Jr. PAS domain allostery and light-induced conformational changes in photoactive yellow protein upon I2 intermediate formation, probed with enhanced hydrogen/deuterium exchange mass spectrometry. J. Mol. Biol. 363:148, 2006.

Panda, K., Haque, M.M., Garcin-Hosfield, E.D., Durra, D., Getzoff, E.D., Stuehr, D.J. Surface charge interactions of the FMN module governs catalysis by nitric-oxide synthase. J. Biol. Chem., in press.

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

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. 280:39208, 2005.

Wood, T.I., Barondeau, D.P., Hitomi, C., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis. Biochemistry 44:16211, 2005.