The Skaggs Institute
for Chemical Biology

Insights Into Protein Chemistry and Biology From Protein Structure

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

We investigate the chemistry and biology of proteins, starting from the determination and analysis of protein structures. In projects funded by the Skaggs Institute, we have focused on understanding catalysis and regulation of the redox-active superoxide dismutase and nitric oxide synthase (NOS) metalloenzymes that control reactive oxygen species and on protein-cofactor interactions that regulate the response of photoactive proteins to light. We integrate high-resolution crystallographic results with those from spectroscopy, hydrogen-deuterium exchange mass spectrometry, and x-ray scattering to probe chemical, conformational, and dynamic changes in proteins and their cofactors. On the basis of our integrated results, we propose comprehensive mechanistic models that explain how proteins function as efficient catalysts and molecular machines. We test these hypotheses with biochemical and mutational analyses, to improve understanding of how proteins achieve and regulate their activities and to aid applications of this knowledge for the design of proteins and inhibitors.

This year, we achieved major advances in isozyme-selective inhibition of NOS, including the development of the anchored plasticity approach for the design of selective inhibitors. Our research on the light-activated DNA-repair of (6-4) photoproducts by the enzyme (6-4) photolyase also shed light on how human cryptochromes function in circadian clocks to control biological rhythms.

Isozyme-Selective Inhibition of Nitric Oxide Synthase

The 3 human NOS isozymes offer key therapeutic targets for neurotransmission (neuronal NOS), regulation of blood pressure (endothelial NOS), and the immune response (inducible NOS). These highly similar, but differently regulated, isozymes all synthesize the diatomic molecule nitric oxide, which is both a molecular signal (at low concentrations) and a cytotoxin (at high concentrations). The aims of our ongoing cross-disciplinary mutational, biochemical, and structural investigations of NOS are to (1) determine the bases for functional domain interactions, cofactor recognition, and tuning for electron transfer and catalysis; (2) characterize the diverse regulatory mechanisms that differentially control the NOS isozymes; and (3) elucidate distinguishing features for isozyme-specific inhibitors. Isozyme-specific NOS inhibitors are sought for medicinal purposes and for advancing understanding of basic human physiology but present a huge challenge because of active-site conservation.

Our comprehensive structural and mutagenesis analyses of NOS in complexes with isozyme-selective inhibitors revealed determinants for isozyme selectivity. In inducible but not endothelial NOS, bulky inhibitors promote a cascade of conformational changes up to 20 Å away from the substrate and inhibitor-binding site (Fig. 1). Correlated side-chain rotations accommodate the rigid bulky tails of the selective inhibitors and expose a new specificity pocket for enhanced inhibitor binding in inducible NOS. Although first-shell (touching the inhibitor) and second-shell (touching the first shell) residues that begin this conformational cascade are invariant, in endothelial NOS their correlated rotations leading to the opening of the specificity pocket are precluded by bulky isozyme-specific residues at the far end. Thus, isozyme differences in the plasticity of second- and third-shell residues modulate conformational changes of invariant first-shell residues to determine inhibitor selectivity.

Fig. 1. Isozyme-selective inhibition of NOS. In both isozymes, the aminopyridine core of the inhibitor (orange) stacks above the heme and forms hydrogen bonds (black dots) mimicking those of the arginine substrate. In human endothelial NOS (left), the long tail of the inhibitor protrudes between the heme carboxylate groups (foreground). In inducible NOS (right), the inhibitor tail projects upward, inducing a cascade of side-chain conformational changes leading to the opening of a new selectivity pocket that enhances binding affinity. In endothelial NOS, conformational changes in the conserved glutamine (Q246) and arginine (R249) side chains are prevented by bulky isozyme-specific amino acids distant from the substrate-binding pocket.

Our combined results allowed us to propose, and successfully test, the anchored plasticity approach for the design of selective inhibitors. With this approach, we exploit conserved binding sites coupled to distant isozyme-specific residues via cascades of conformational changes; the inhibitor core is designed to mimic the binding of a substrate or cofactor in a conserved binding site. This anchor is extended by rigid bulky substituents oriented along pathways leading to sequence or structural variations needed for selectivity. This anchored plasticity approach exemplifies general principles for the development of novel selective inhibitors that overcome active-site conservation.

Protein Partnership with FAD to Repair DNA or Control Biological Clocks

We are investigating how living things use cofactor-protein partnerships to transduce environmental changes into appropriate biological responses. Proteins of the cryptochrome/photolyase family share not only the same protein fold but also the redox-active FAD cofactor bound in an unusual U-shaped conformation beneath a positively charged groove designed for DNA binding. Through structural and functional studies of diverse members of the cryptochrome/photolyase families, we are deciphering how the similarities and differences in these molecules direct the same cofactor and protein fold to produce different biological responses to light: cryptochromes control biological rhythms, whereas photolyases repair DNA damage.

To develop and test hypotheses for structure-function relationships in the cryptochrome/photolyase family, we determined the x-ray crystallographic structure of (6-4) photolyase, which confers UV protection to plants and has high sequence similarities (~50% identity) to human cryptochromes (Fig. 2). In humans and other vertebrates, cryptochromes are essential components of the circadian clock, which regulates sleep-wake cycles and other daily biological rhythms. The eukaryotic (6-4) photolyase structure revealed a substrate recognition site specific for the UV-induced DNA lesion, the (6-4) photoproduct, and cofactor binding sites different from those of the bacterial photolyase, consistent with distinct mechanisms for activities and regulation. The entrance to the active-site cavity above FAD is constricted by adjacent phosphate-binding and protrusion motifs that correlate with a phosphorylation site and nuclear localization sequence for cryptochrome. We coupled our structural studies with site-directed mutagenesis and functional assays. We tested (6-4) photolyase mutants for DNA-repair activity and tested mouse cryptochrome mutants for clock functions by transient transfection assays done in collaboration with L. DeHaro and S. Panda, the Salk Institute for Biological Studies.

Fig. 2. Structure of (6-4) photolyase. Ribbon diagram shows the N-terminal αβ domain (top), the helical domain with bound FAD cofactor (yellow), and the C-terminal extension (lower right) for a eukaryotic photolyase that also serves as an improved model for human cryptochromes.

Publications

Biskup, T., Schleicher, E., Okafuji, A., Link, G., Hitomi, K., Getzoff, E.D., Weber, S. Direct observation of a photoinduced radical pair in a cryptochrome blue-light photoreceptor. Angew. Chem. Int. Ed. 48:404, 2009.

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. 4:700, 2008.

Shin, D.S., Didonato, M., Barondeau, D.P., Hura, G.L., Hitomi, C., Berglund, J.A., Getzoff, E.D., Cary, S.C., Tainer, J.A. Superoxide dismutase from the eukaryotic thermophile Alvinella pompejana: structures, stability, mechanism, and insights into amyotrophic lateral sclerosis. J. Mol. Biol., in press.