News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
Principles of Protein Structure for Chemical Recognition, Complementarity,
E.D. Getzoff, M. Aoyagi, A.S. Arvai, R.M. Brudler, C.M. Bruns, B.R. Crane,
K.T. Forest, U.K. Genick, T.P.K. Lo, S.E. Mylvaganam, J.L. Pellequer, M.E. Pique,
R.J. Rosenfeld, M.E. Stroupe, M.M. Thayer, M.J. Thompson
We study the structural chemistry underlying protein molecular recognition,
complementarity, and catalysis at the atomic level. In these problem-directed
studies, we use molecular biology, biochemistry, x-ray crystallography, and spectroscopy
together with computational and computer graphics analysis to characterize the
structural chemistry and function of proteins. We then test our understanding
by protein design. State-of-the-art technologies we use include multiwavelength
anomalous diffraction, time-resolved Laue crystallography, femtosecond and nanosecond
laser initiation, and rapid spectroscopic characterization. Taken together, these
methods enable us to provide a detailed understanding of how proteins work, rather
than simply supply a description of protein structures.
Biocatalysis of Sulfur Transformations
Sulfite and nitrite reductases catalyze fundamental chemical transformations
for biogeochemical cycling of sulfur and nitrogen. We used multiwavelength anomalous
diffraction of the native siroheme and Fe4S4 cluster cofactors
to solve the atomic resolution structure of sulfite reductase hemoprotein, which
catalyzes the concerted 6-electron reductions of sulfite to sulfide and nitrite
to ammonia. We determined 12 key high-resolution structures of sulfite reductase
hemoprotein that characterize its active center in 3 different states of oxidation
and its interactions with substrates, inhibitors, intermediates, and products.
Coupled crystallographic and spectroscopic studies revealed the mechanism for
heme activation via reduction-gated exogenous ligand exchange and provided descriptions
of the intermediates at each step along the complex reaction pathway (Fig. 1).
Oxygen Recognition and Reactive Oxygen Catalysis
Ongoing research in the Getzoff and Tainer laboratories aims to understand
the unique structural metallobiochemistry of nitric oxide synthase (NOS) and
superoxide dismutase (SOD), which produce and regulate reactive oxygen species.
Cu,Zn SOD, which dismutes superoxide anions into oxygen and hydrogen peroxide,
is a master eukaryotic regulator of reactive oxygen species, because most free
radicals are scavenged by dioxygen to superoxide. Biochemically, eukaryotic Cu,Zn
SODs are remarkable for their unusually great subunit and dimer stability, faster-than-diffusion
attraction of substrate, exquisite specificity, and efficient catalysis. Biologically,
SODs are important for their antioxidant, antiinflammatory, and antiaging properties.
Moreover, by controlling levels of superoxide available for rapid reaction with
nitric oxide to form peroxynitrite, SODs also regulate nitric oxide, an important
biological signal and defensive cytotoxin (see following). Medically, genetic
mutations in human Cu,Zn SOD cause the fatal degenerative disease of motor neurons
termed amyotrophic lateral sclerosis or Lou Gehrig disease.
Our new structures of bacterial Cu,Zn SODs from symbionts and pathogens provide
the potential for drug design. Whereas the fold and active-site geometry of bacterial
Cu,Zn SODs match those of eukaryotic SODs, the elements recruited to form the
dimer interface and the active-site channel are strikingly different. Our structures
and redesign of SOD aim to elucidate the structural metallobiochemistry and structure-function
relationships of the enzyme.
Our new research on the structural and chemical biology of NOS, which regulates
the synthesis and thereby the biological activity of nitric oxide, complements
our studies on SOD. Nitric oxide functions at low concentrations as a diffusible,
biological messenger for neurotransmission, long-term potentiation, platelet
aggregation, and regulation of blood pressure. At higher concentrations, nitric
oxide acts as a cytotoxic agent for defense against tumor cells and intracellular
Each NOS subunit is divided into 2 domains joined by a calmodulin-binding
hinge region: (1) an oxygenase domain with binding sites for heme, tetrahydrobiopterin,
and substrate that forms the catalytic center for production of nitric oxide
and (2) a reductase domain with binding sites for NADPH, FAD, and FMN that supplies
electrons to the oxygenase domain. We crystallized both domains and determined
refined structures for the oxygenase domain, in monomeric and dimeric forms,
and in complex with cofactors, substrate, and inhibitors. Our structures revealed
a novel protein fold and characterized the roles of the cofactors, and of the
l-arginine substrate itself, in the 2 steps of the reaction mechanism. First,
l-arginine is oxidized to N-hydroxy-l-arginine by a monooxygenase-like reaction;
then this intermediate is oxidized to form citrulline and nitric oxide by an
Chemical Biology of PAS Domains of Sensors and Clock Proteins
We aim to characterize the structural and chemical biology of PER-ARNT-SIM
(PAS) domains (Fig. 2). These domains were originally identified as sequence
repeats in the Drosophila clock protein PER and the basic helix-loop-helix--containing
transcription factors aryl hydrocarbon receptor nuclear translocator (ARNT) in
mammals and single-minded (SIM) in flies.
PAS domain sequences, which mediate protein-protein interaction and in some
cases bind small ligands, also occur in sensor kinase proteins, the phytochrome
photoreceptors of plants, and the newly discovered eukaryotic clock proteins,
including the clock proteins that clearly couple photoreception to circadian
rhythms. PAS domain sequences show similarities to photoactive yellow protein,
a bacterial blue-light photosensor whose structural molecular biology has been
under study. The aim of research funded by the Skaggs Institute is to use our
0.83-Å resolution structure of photoactive yellow protein to determine
structures for PAS domains and to characterize the roles of these domains in
Coupled Crystallographic and Spectroscopic Analysis of Protein Mechanisms
The purpose of our project on coupled crystallographic and spectroscopic
analysis of protein mechanisms is to establish new state-of-the-art technology
at The Scripps Research Institute and provide leadership in a key area of chemical
structural biology. The laser and spectroscopy facility that we established in
this laboratory enables us to initiate and spectroscopically monitor reactions
in protein crystals on a timescale of nanoseconds. This facility provides the
opportunity for detailed characterization of the many interesting transient structural
states of proteins responsible for catalysis and signaling, which typically have
life-times shorter than milliseconds. Rapid monitoring of spectroscopic properties
in protein crystals allows determination of the precise oxidation state of metal
centers, specific assays of function in the crystalline state, and the ability
to establish conditions that allow sufficient accumulation of transient intermediates
for structure determination by time-resolved crystallography.
Crane, B., Siegel, L., Getzoff, E.D. Probing the catalytic mechanism
of sulfite reductase by x-ray crystallography: Structure of the Eschericia
coli hemoprotein in complex with substrates, inhibitors, intermediates and
products. Biochemistry 36:12120, 1997.
Crane, B., Siegel, L., Getzoff, E.D. Structures of the siroheme and
Fe4S4-containing active center of sulfite reductase in
different states of oxidation: Heme activation via reduction-gated exogenous
ligand exchange. Biochemistry 36:12101, 1997.
Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D.,
Stuehr, D.J., Tainer, J.A. The structure of nitric oxide synthase oxygenase
domain and inhibitor complexes. Science 278:425, 1997.
Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C., D.K., Getzoff, E.D., Stuehr,
D.J., Tainer, J.A. Structure of nitric oxide synthase oxygenase dimer with
pterin and substrate. Science 279:2121, 1998.
Devanathan, S., Genick, U.K., Canestrelli, I.L., Meyer, T.E., Cusanovich,
M.A., Getzoff, E.D., Tollin, G. New insights into the photocycle of Ecotothiorhodospira
halophila photoactive yellow protein: Photorecovery of the long-lived photobleached
intermediate of the Met100Ala mutant. Biochemistry, in press.
Fisher, C.L., Cabelli, D.E., Hallewell, R.A., Beroza, P., Lo, T.P., Getzoff,
E.D., Tainer, J.A. Computational, pulse-radiolytic, and structural investigations
of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide
dismutase. Proteins 29:103, 1997.
Genick, U.K., Soltis, S.M., Kuhn, P., Canestrelli, I.L., Getzoff, E.D., Structure
at 0.85-Å resolution of an early protein photocycle intermediate. Nature
Mylvaganam, S.E., Paterson, Y., Getzoff, E.D. Structural basis for
the binding of an anti-cytochrome c antibody to its antigen: Crystal structures
of FabE8-cytochrome c complex to 1.8-Å resolution and FabE8 to 2.26-Å resolution.
J. Mol. Biol., in press.
Pellequer, J.L., Wager-Smith, K.A., Kay, S.A., Getzoff, E.D. Photoactive
yellow protein: A structural prototype for the three-dimensional fold of the
PAS domain superfamily. Proc. Natl. Acad. Sci. U.S.A., in press.