Scientific Report 2008
Protein Structures, Activities, and Regulation:
The Functioning of Molecular Machines
E.D. Getzoff, A.S. Arvai, E.D. Garcin,
C. Hitomi, K. Hitomi, M.D. Kroeger, M.E. Pique, A.J. Pratt, D.S. Shin
how proteins function as molecular machines, we use structural molecular biology
to characterize and control proteins. We apply the tools of structural, molecular,
and computational biology to analyze proteins of biological and biomedical interest,
especially proteins that work synergistically with coupled chromophores, metal ions,
or other cofactors.
Nitric Oxide Synthase and
a New Approach to Design of Selective Inhibitors
Nitric oxide synthase (NOS) enzymes synthesize
nitric oxide, a signal for vasodilation and neurotransmission at low levels and
a defensive cytotoxin at higher levels. Synthesis of nitric oxide by NOS requires
calmodulin-coordinated interactions between the catalytic, oxygenase,
and electron-supplying reductase modules of the enzyme. NOS uses heme, zinc, tetrahydrobiopterin,
calcium, NADPH, FMN, and FAD cofactors.
The goals of our structure-based drug
design projects are to selectively inhibit inducible NOS, to prevent inflammatory
disorders, or neuronal NOS, to prevent migraines, while maintaining blood pressure
regulation by endothelial NOS. Our x-ray crystallographic structures of wild-type
and mutant NOS oxygenase dimers with substrate, intermediate, inhibitors, cofactors,
and cofactor analogs, determined in collaboration with J.A. Tainer, Department
of Molecular Biology, and D. Stuehr, the Cleveland Clinic, Cleveland, Ohio,
provide insights into both catalytic mechanism and inhibitor selectivity. The nearly
complete sequence and structural conservation in the active sites of the 3 NOS isozymes
is a significant challenge in the design of isozyme-specific inhibitors. Nevertheless,
our latest results indicate that plasticity of distant isozyme-specific residues
modulates conformational changes of invariant residues in the substrate-binding
site. We applied these results to develop the anchored plasticity approach for the
structure-based design of selective inhibitors (Fig. 1). In this approach, conserved
binding sites are used to anchor the core of an inhibitor, while distant sequence
differences are exploited to provide selectivity.
Diagram illustrating the anchored-plasticity approach to selective drug design.
Our structure of the neuronal NOS reductase
reveals new insights into the complex regulatory mechanisms of this enzyme family.
We integrated biochemical data with our structures of NOS oxygenase, NOS reductase,
and calmodulin in complex with NOS peptides to propose assembly and mechanistic
hypotheses for the holoenzyme. We obtained promising results in support of these
hypotheses by using solution small-angle x-ray scattering, which can provide molecular
envelopes for macromolecules and macromolecular complexes in solution.
We have proposed a moving-domain mechanism
for controlling the rate-limiting flow of electrons from the flavin cofactors of
NOS reductase to the catalytic NOS oxygenase heme. Our assembly and mechanistic
hypotheses also explain the kinetics of regulatory site-specific phosphorylation
and dephosphorylation events that both activate and inactivate synthesis of nitric
oxide in vivo. In ongoing research, we are using complementary biochemical, biophysical,
and computational methods to define, describe, and understand how NOS chemistry,
structure, assembly, dynamics, and protein-protein interactions regulate production
of nitric oxide in vivo.
Photoactive Proteins and Circadian Clocks
To understand in atomic detail how chromophore-bound
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 FAD-containing proteins
of the photolyase/cryptochrome family catalyze DNA repair or act in circadian clocks.
To understand the photocycle of PYP and to propose a common mechanism for signaling
by Per-Arnt-Sim domains, we combined ultra-high-resolution and time-resolved crystallographic
structures of the PYP dark state and 2 photocycle intermediates with site-directed
mutagenesis; spectroscopy; deuterium-hydrogen exchange mass spectrometry, in collaboration
with V. Woods, University of California, San Diego; and quantum mechanical and electrostatic
computational methods, in combination with L. Noodleman, Department of Molecular
flavoproteins function as blue-light receptors in plants and as components of circadian
clocks in animals. We determined the first crystallographic structure of a cryptochrome;
the structure revealed commonalities with the homologous photolyases in DNA binding
and redox-dependent function but showed differences in active-site and interaction-surface
features. We found that this cryptochrome binds he same antenna cofactor found in a photolyase
homolog but uses different amino acid residues to form the cofactor-binding site.
Our new structures and spectroscopy, obtained in collaboration with S. Weber, Universität
Freiburg in Germany, of cryptochromes and of photolyases from 2 other branches of
the photolyase/cryptochrome family that repair cyclobutane pyrimidine dimers
and (6-4) photoproducts help us decipher the cryptic structure, function, and evolutionary
relationships of these fascinating redox-active proteins. Furthermore, the (6-4)
photolyase enzyme provides an excellent model for human cryptochrome.
A simple, but functional, circadian clock
can be reconstituted in vitro from the 3 cyanobacterial proteins KaiA, KaiB, and
KaiC alone. Yet, the structure and dynamics of the functional assembly are not understood.
Our crystallographic, dynamical light scattering and small-angle x-ray scattering
studies revealed that KaiB self-assembles into a tetramer. We also study clock proteins
with PYP-like 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 and
regulate biological function.
The superoxide radical is a central player
in the biology of reactive oxygen and nitrogen intermediates, which mediate signaling
and oxidative damage, a key factor in aging and cancer, in vivo. Mutations in human
copper zinc superoxide dismutase (SOD), the enzyme that converts superoxide to molecular
oxygen and hydrogen peroxide, cause the fatal neurodegenerative disease familial
amyotrophic lateral sclerosis, or Lou Gehrig disease. We are analyzing the structural
chemistry of SOD to help bridge the gap from protein structures to enzyme stability
and activities in vivo. We designed a zinc-free variant of human SOD to help test
the role of zinc binding and loss in disease. Our results, obtained in collaboration
with J.A. Tainer, support the importance of the stable SOD core structure in preventing
amyloid formation and toxic effects. For comparison, we determined the structure
and stability of the SOD from the most extreme eukaryotic thermophile known: the
deep-sea hydrothermal vent worm Alvinella pompejana.
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., in press.
B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., Anderson, S.R., Bomben, V.C.,
Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural
characterization of zinc-deficient human superoxide dismutase and implications for
ALS. J. Mol. Biol. 373:877, 2007.
Yamamoto, J., Tanaka, Y., Hitomi,
K., Getzoff, E.D., Iwai, S.
Spectroscopic studies on a novel intramolecular hydrogen bond within the (6-4) photoproduct.
Nucleic Acids Symp. Ser. (Oxf). Issue 51:79, 2007.