The Skaggs Institute
for Chemical Biology

PYP is the prototype for the ubiquitous Per-Arnt-Sim (PAS) domains of proteins that mediate intraprotein and interprotein interactions and conformational changes in response to light, oxygen, redox potential, and small-molecule ligands. In collaboration with V. Woods, University of California, San Diego, we used enhanced hydrogen-deuterium exchange mass spectrometry to probe the conformational and dynamic changes that occur in PYP in solution upon light activation. Our results revealed that upon light activation, the central β-sheet of PYP remains stable, but disruption of the dark-state hydrogen-bonding network produces increased flexibility and opening of PAS core helices (α3 and α4), releases the β-hairpin (linking strands β4 and β5), and propagates conformational changes to the central β-sheet (Fig. 1). Surprisingly, approximately the first 10 N-terminal residues, which are essential for fast return to the dark state, become more protected in the long-lived, light-activated intermediate. By integrating our results with those from diverse biophysical studies, we propose a versatile signal transduction mechanism that links an allosteric T(tense)-to-R(relaxed) state conformational transition to 3 pathways (arrows, Fig. 1) for signal propagation within the structurally conserved PYP/PAS fold.

Fig. 1. Signal transduction in the PYP/PAS fold. Central ribbon representation shows the most significant changes in deuterium-hydrogen exchange upon light activation of PYP. Arrows indicate proposed pathways for signal transduction pathways (through the PAS core helices, the β-hairpin, or the β-sheet interface) for framework-encoded allostery in the PYP/PAS module. Surrounding plots show deuterium-hydrogen exchange kinetics for representative segments of PYP.

For green and red fluorescent proteins, our high-resolution crystallographic structures are providing important new insights into the mechanisms by which 3 sequential component amino acid residues undergo spontaneous posttranslational modifications to form their striking fluorescent chromophores. We have determined structures of trapped intermediates in fluorophore biosynthesis, proposed a novel conjugation-trapping mechanism, and identified protein features key to chromophore formation and spectral tuning. We discovered that the specific features of the architecture of green fluorescent protein that favor the remarkable peptide cyclization and oxidation reactions for fluorophore biosynthesis can also drive diverse alternative chemistries, including an array of peptide hydrolysis and cleavage reactions (Fig. 2).

Fig. 2. Cleavage sites favored by the protein architecture and chemistry of red and green fluorescent proteins. Arrows indicate bonds that are spontaneously cleaved in homologs and mutants of green fluorescent protein.

We identified and characterized the CryDASH cryptochrome protein family, which occurs in animal, plant, and bacterial species (in contrast with classic animal- and plant-specific cryptochrome families). We determined the first cryptochrome crystallographic structure, which showed homology with the photolyase family of light-active DNA repair proteins. Cryptochromes share with photolyases not only the overall protein fold but also the redox-active FAD cofactor bound in an unusual U-shaped conformation and the surrounding positive electrostatic surface consistent with a function in DNA binding. Through structural and functional studies of diverse members of the cryptochrome/photolyase families, we are deciphering how their similarities and differences direct the same cofactor and protein fold to produce different biological responses to light.

Chemical Biology and Regulation of Nitric Oxide Synthases

We continue to use Skaggs funding for investigations of nitric oxide synthases (NOSs), which are key therapeutic targets for neurotransmission (neuronal NOS), blood pressure regulation (endothelial NOS), and the immune response (inducible NOS). These 3 similar, but differently regulated, isozymes all synthesize the diatomic molecule nitric oxide, which is paradoxically both a molecular signal (at low concentrations) and a cytotoxin (at high concentrations). Isozyme-specific NOS inhibitors are highly desirable for medicinal purposes and for advancing understanding of basic human physiology but present a huge challenge. Each of the 3 NOS isozymes has 2 major modules: (1) a catalytic oxygenase module with binding sites for heme, tetrahydrobiopterin, and substrate and (2) an electron-supplying reductase module with binding sites for NADPH, FAD, and FMN. The 2 modules are covalently connected via a central linker that binds the calcium-regulated protein calmodulin.

We have determined a series of crystallographic structures to define the oxygenase, the reductase, and the calmodulin-bound linker; characterize their cofactor binding; and identify mechanisms for their functions in synthesis and regulation of nitric oxide. In collaboration with J.A. Tainer, the Skaggs Institute, we are using small-angle x-ray scattering to test our assembly and mechanistic models for NOS by defining the shapes and conformational changes of NOS domains and their assemblies. In our recent crystallographic efforts, we focused on analyzing the isozyme-specific mechanisms used to selectively bind different classes of inhibitors.

The aims of these ongoing integrated cross-disciplinary mutational, biochemical, and structural investigations of NOS are to (1) determine the basis for functional domain interactions, cofactor recognition, and how the NOS enzymes tune these cofactors to perform complex catalytic and redox chemical reactions; (2) identify and apply the features that distinguish among the 3 isozymes to the design of isozyme-specific inhibitors; and (3) understand the diverse regulatory mechanisms that differentially control the NOS isozymes.

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 govern catalysis by nitric-oxide synthase. J. Biol. Chem. 281:36819, 2006.

Schleicher, E., Hitomi, K., Kay, C.W., Getzoff, E.D., Todo, T., Weber, S. ENDOR differentiates complementary roles for active site histidines in (6-4) photolyase. J. Biol. Chem., in press.