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
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.
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.
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
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.
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.