The Skaggs Institute
for Chemical Biology
Scientific Report 2005
P.E. Dawson, F. Brunel,
M. Churchill, N. Metanis, T. Tiefenbrunn
on using synthetic chemistry to understand the molecular basis of protein structure and function.
Total chemical synthesis facilitates the fine-tuning of amino acid side chains to achieve, for
example, greater conformational stability or to alter the binding specificity of proteins. Currently,
we are developing therapies and potential vaccine candidates for HIV disease and are doing research
to understand the molecular basis of enzymatic catalysis.
Glutaredoxin is an oxidoreductase with
homology to the thioredoxins and protein disulfide isomerases. These oxidoreductases are critical
regulators of redox potential and the formation of disulfide bonds in cells. Oxidoreductases
use a conserved CXXC motif to tune the redox potential of the active site. Glutaredoxin functions
as a hydride donor for ribonuclotide reductase. We are using chemical synthesis to introduce the
amino acid selenocysteine into the active site of glutaredoxin to investigate the effects of forming
sulfur-selenium or selenium-selenium bonds on the redox potential of this protein. In addition,
we anticipate that the mixed selenium-sulfur enzymes will gain significant peroxidase activities.
Using chemical ligation strategies, we have now synthesized a series of glutaredoxin analogs,
and we are characterizing their structures and functions.
HIV Vaccine Design
Eliciting broadly neutralizing antibodies
is a major goal in HIV vaccine design. This effort is complicated by the poor accessibility of conserved
regions of HIV envelope proteins to antibodies. The membrane proximal region of the HIV envelope
protein gp41 has generated marked research because of the discovery of 2 neutralizing antibodies
that bind this sequence. We are collaborating with D.R. Burton, Scripps Research, and I.A. Wilson,
the Skaggs Institute, to design peptides that mimic the helical conformation of the peptide that
binds to the neutralizing antibody 4E10.
Immunization with simple peptides obtained
from gp41 elicit a nonneutralizing response, presumably because of differences between the neutralizing
and the most immunogenic conformations of the peptide. We fully characterized the 4E10 epitope
and constrained the peptide to adopt the primarily helical conformation recognized by the 4E10
antibody. These studies have yielded a series of peptidomimetics that bind as tightly to the 4E10
antibody as the full-length gp41 glycoprotein does. Our goal is to eliminate all surfaces of the
peptide that are not required for 4E10 binding or to mask them with soluble polymers and carbohydrates.
We plan to use this negative design approach in combination with binding studies, immunization,
and structural analysis by crystallography.
Glycosylation of the HIV coat proteins
is critical for viral stability and protection of the virus from the immune system. Recent research
indicated that rare oligosaccharides rich in mannose on this virus are involved in molecular interactions
with human proteins such as DC-SIGN that are critical for HIV infection. These oligosaccharides
are recognized by HIV-neutralizing proteins from cyanobacteria; one of these proteins, cyanovirin,
is in clinical trials as an HIV microbicide. We are developing a total synthesis of the gp120-binding
proteins DC-SIGN and cyanovirin to enable detailed structure-function studies on their carbohydrate-binding
properties and their potential as HIV-inhibiting pharmaceuticals. In particular, efforts will
be directed toward improving affinity and stability. Finally, we are interested in developing
polyvalent dendrimers of these domains to increase multivalent interactions with the HIV coat
Beltran, A.C., Dawson, P.E., Gottesfeld,
J.M. Role of DNA sequence in the binding specificity of synthetic
basic-helix-loop-helix domains. Chembiochem 6:104, 2004.
Brunel, F.M., Dawson, P.E. Synthesis
of constrained helical peptides by thioether ligation: application to analogs of gp41. Chem.
Commun. (Camb.) 2552, 2004, Issue 20.
Deechongkit, S., Dawson, P.E.,
Kelly, J.W . Toward assessing the position-dependent contributions
of backbone hydrogen bonding to β-sheet
folding thermodynamics employing amide-to-ester perturbations. J. Am. Chem. Soc. 126:16762,
Kamikubo, Y., De Guzman, R., Kroon,
G., Curriden, S., Neels, J.G., Churchill, M.J., Dawson, P., Oldziej, S., Jagielska, A., Scheraga,
H.A., Loskutoff, D.J., Dyson, H.J. Disulfide bonding arrangements
in active forms of the somatomedin B domain of human vitronectin. Biochemistry 43:6519, 2004.
McNulty, J.C., Jackson, P.J., Thompson,
D.A., Chai, B., Gantz, I., Barsh, G.S., Dawson, P.E., Millhauser, G.L.
Structures of the agouti signaling protein. J. Mol. Biol. 346:1059, 2005.
Metanis, N., Keinan, E., Dawson
P.E. A designed synthetic analog of 4-OT is specific for a
non-natural substrate. J. Am. Chem. Soc.127:5862, 2005.