News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
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,
M. DiDonato, K.T. Forest, E.D. Garcin, U.K. Genick, C.K. Koike, S.J. Lloyd, 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 and inhibitor design. State-of-the-art technologies we use include
multiwavelength anomalous diffraction, time-resolved Laue crystallography, ultra-high-resolution
protein 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, 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 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.
Oxygen Recognition and Reactive Oxygen Catalysis
Ongoing research in the Getzoff and Tainer laboratories aims to understand
the unique structural metallobiochemistry of nitric oxide synthases (NOSs) and
superoxide dismutases (SODs), which produce and regulate reactive oxygen species.
Cu,Zn SOD, which dismutes superoxide anions into oxygen and hydrogen peroxide,
is an antioxidant enzyme and a master eukaryotic regulator of reactive oxygen
species. 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). Biochemically,
eukaryotic Cu,Zn SODs are remarkable for their unusually great stability, faster-than-diffusion
attraction of substrate, exquisite specificity, and efficient catalysis. Biologically,
SODs are important for their antioxidant, antiinflammatory, and antiaging properties.
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 (Fig. 1).
Our structures and redesign of SODs aim to elucidate the structural metallobiochemistry
and structure-function relationships of the enzymes.
Our new research on the structural and chemical biology of NOSs, which regulate
the synthesis and thereby the biological activity of nitric oxide, complements
our studies on SODs. 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 heme. We crystallized both domains and determined refined
structures for the oxygenase domain, in monomeric and dimeric forms, and in complex
with cofactors, cofactor analogs, substrate, intermediate, 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 unprecedented reaction. Ongoing structural and computational studies of
NOS-inhibitor complexes are providing an interactive design cycle for the structure-based
design of optimized inhibitors.
Chemical Biology of Per-Arnt-Sim Domains of Sensors and Clock Proteins
We aim to characterize the structural and chemical biology of PER-ARNT-SIM
(PAS) domains. These domains were originally identified as sequence repeats in
the Drosophila clock protein PER and the basic-helix-loop-helixcontaining
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. The structures of PAS domains
resemble the structure of photoactive yellow protein (Fig. 2), 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 signal transduction. The results
will provide the basis to ultimately control PAS domain sensors in plants and
animals for numerous applications in biotechnology.
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 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.
Our coupled crystallographic and spectroscopic results will lead to understanding
and ultimately control of reaction pathogenesis in enzymes and signal transduction
Crane, B.R., Rosenfeld, R.J., Arvai, A.S., Ghosh, D.K., Ghosh, S., Tainer,
J.A., Stuehr, D.J., Getzoff, E.D. N-terminal domain swapping and metal ion
binding in nitric oxide synthase dimerization. EMBO J., in press.
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 33:11563, 1998.
Ghosh, D.K., Crane, B.R., Ghosh, S., Wolan, D., Gachhui, R., Crooks, C.,
Presta, A., Tainer, J.A., Getzoff, E.D., Stuehr, D.J. Inducible nitric oxide
synthase: Role of the N-terminal ß-hairpin hook and pterin-binding segment
in dimerization and tetrahydrobiopterin interaction. EMBO J., in press.
Lo, T.P., Thayer, M.M., Koike, C.K., Hallewell, R.A., Getzoff, E.D., Tainer,
J.A. Variability among multiple subunits in the high-resolution structure
of human Cu,Zn superoxide dismutase. In: Superoxide Dismutase: Recent
Advances and Clinical Applications. Edeas, M.A. (Ed.). Editions Mel Paris, Paris,
France, 1999, p. 22
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. 281:301, 1998.
Pellequer, J.-L., Brudler, R., Getzoff, E.D. Biological sensors: More
than one way to sense oxygen. Curr. Biol. 9:R416, 1999.
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. 95:5884, 1998.