Molecular Biology - The Scripps Research Institute

168 MOLECULAR BIOLOGY 2005
Fig. 2. The tetrameric assembly of the cyanobacterial circadian
clock protein KaiB revealed by small-angle x-ray scattering (experimentally
determined shape) and x-ray crystallography (ribbon showing
protein fold).
teins, define their mechanisms of action and interaction,
and use our results to understand and regulate
biological function.
METALLOENZYME STRUCTURE AND FUNCTION
Superoxide dismutases (SODs) act as master regulators
of intracellular free radicals and reactive oxygen
species by transforming superoxide to oxygen and
hydrogen peroxide. Novel nickel SODs assemble into
hollow spheres composed of six 4-helix bundle subunits.
The 9 N-terminal residues fold into a unique
nickel hook motif that shows promise as a detectable
metal ion–binding tag in protein purification and structure
determination.
Our crystallographic structures of classic copper-zinc
SODs from mammals, bacterial symbionts, and pathogens
revealed striking differences in the enzyme assembly
and in the loops flanking the active-site channel,
despite the shared β-barrel subunit fold, catalytic
metal center, and electrostatic enhancement of activity.
With J. Tainer, Department of Molecular Biology,
we determined structures of mutant human SODs
found in patients with the disease amyotrophic lateral
sclerosis (Lou Gehrig disease), and proposed a hypothesis
for how single-site mutations cause this fatal neurodegenerative
disease.
To synthesize nitric oxide, a cellular signal and defensive
cytotoxin, nitric oxide synthases (NOSs) require calmodulin-orchestrated
interactions between their catalytic,
heme-containing oxygenase module and their electronsupplying
reductase module. Crystallographic structures
Published by TSRI Press ®. ©Copyright 2005,
The Scripps Research Institute. All rights reserved.
of wild-type and mutant NOS oxygenase dimers with
substrate, intermediate, inhibitors, cofactors, and cofactor
analogs, determined in collaboration with D. Stuehr,
the Cleveland Clinic, Cleveland, Ohio, and J. Tainer,
provided insights into the catalytic mechanism and
dimer stability.
Our structure-based drug design projects are aimed
at selectively inhibiting inducible NOS, to prevent inflammatory
disorders, or neuronal NOS, to prevent migraines,
while maintaining blood pressure regulation by endothelial
NOS. We integrated biochemical data with our
structures of NOS oxygenase, NOS reductase, and calmodulin
in complex with peptides derived from NOS
to propose a model for the assembled holoenzyme that
provides a moving-domain mechanism for electron flow
from NADPH through 2 flavin cofactors to the heme.
Our structure of the NOS reductase provides new
insights into the complex regulatory mechanisms of
this enzyme family.
METALLOPROTEIN DESIGN
An ultimate goal for protein engineers is to design
and construct new protein variants with desirable catalytic
or physical properties. As members of the Scripps
Research Metalloprotein Structure and Design Group,
we are testing our understanding of the affinity, selectivity,
and activity of metal ions by transplanting metal
sites from structurally characterized metalloproteins into
new protein scaffolds. To aid our design efforts, we have
organized quantitative information and interactive viewing
of protein metal sites at the Metalloprotein Database
and Browser (available at http://metallo.scripps.edu).
For green fluorescent protein and the homologous
red fluorescent protein, we designed, constructed, and
characterized metal-ion biosensors in which binding of
metal ions is signaled by changes in the spectroscopic
properties of the naturally occurring fluorophores. The
green fluorescent protein scaffold provides advantages
over existing probes by allowing optimization with random
mutagenesis, noninvasive expression in living cells,
and targeting to specific cellular locations. By completing
the metalloprotein design cycle from prediction to
highly accurate structures, we can rigorously evaluate
and improve our algorithms for the design of metal sites.
Our related structural studies of green and red fluorescent
protein intermediates in chromophore cyclization
and oxidation provide a novel mechanism for the spontaneous
synthesis of these tripeptide fluorophores within
the protein scaffold.