News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
Master Keys for Chemical Molecular Biology From Macromolecular Structures
J.A. Tainer, A.S. Arvai, Y. Bourne, C. Bruns, B. Crane, D. Daniels, K.P. Hopfner,
D. Hosfield, T.P. Lo, C. Mol, S. Parikh, C. Putnam, D. Shin
We address central structural questions at the interface of cellular and molecular
biology and chemistry. We wish to characterize and understand protein structures
in order to develop chemical regulators as master keys for controlling fundamental
biological processes associated with reactive oxygen signals and cytotoxins,
pathogenesis, DNA repair, genetic variation, and the cell cycle. Interactions
with staff at the Skaggs Institute have transformed our research by allowing
in-depth characterization of the structure and chemical biology of human enzymes
and initiation of long-term therapeutic goals for new treatments of inflammatory,
degenerative, and infectious diseases; high blood pressure; stroke; and cancer.
Proteins Regulating Genome Stability and Variation
DNA repair proteins balance genome fidelity with the genome variability that
underlies evolutionary selection. Specific mutations leading to decreased genome
stability are critical early events in tumorigenesis, and DNA repair enzymes
are key components in maintaining genome stability. Our new structures of complexes
formed by base-excision repair enzymes and DNA and of complexes consisting of
proteins that directly reverse DNA damage and the removed alkylation damage are
thus providing key insights to the structural chemistry of tumorigenesis relevant
Our structures and kinetics
of wild-type and mutant complexes formed by DNA repair enzymes and damaged DNA
that initiate different pathways of DNA repair suggest new aspects of the structural
chemistry that regulates recognition of DNA damage, progression along the repair
pathway, and avoidance of destructive interference between different DNA repair
steps and pathways. During this past year, for example, we established the mechanism
for recognition and removal of damaged bases by the HhH superfamily of DNA glycosylases
typified by enodnuclease III, which removes oxidized pyrimidines, and MutY, which
removes adenine-guanine mismatches due to oxidized guanine (Fig. 1). Recognition
of damaged bases by these DNA glycosylases appears to provide a structural basis
for specific recruitment of subsequent enzymes that are not recruited by proteins
that act in single-step repair.
Single-step repair is an important type of DNA repair that involves the direct
reversal of damage to DNA bases, such as the reversal accomplished by the alkyltransferases.
Our studies of alkyltransferases typify our structure-based efforts to develop
new cancer treatments that use inhibitors designed to specifically disrupt DNA
repair. O6-alkylguanine alkyltransferase (AGT) is the human DNA repair protein
that removes the promutagenic lesions caused by alkylation of guanine at the
O6 position. Many cancer chemotherapies involve DNA alkylation, and the cytotoxic
effects of the agents used are due to generation of alkylated bases, such as
O6-methylguanine. Because AGT can repair such lesions through a direct and irreversible
alkyl transfer reaction, numerous tumor cell lines develop resistance to alkylation
therapies by increasing expression of AGT. Correspondingly, inhibitors of AGT
can sensitize tumors to alkylation therapies and thereby potentiate current chemotherapies.
To begin an iterative cycle of the structure-based design of inhibitors, we
determined the structure of native human AGT to 2.0-Å resolution. The structure
reveals a 2-domain α/ß protein and identifies the active site. Our
structure of a product complex of AGT with the suicide inhibitor O6-benzylguanine
provides the first look at recognition of damage by the active site of AGT and
the specificity of the active site for damaged areas. Using this complex, the
location of evolutionarily conserved residues, and available kinetic data on
point mutations, we inferred the basis for substrate recognition. AGT presents
a hydrophobic cleft that recognizes alkylated guanine bases with a system of
complementary hydrogen bonds. Comparison of the AGT structure with the structures
of DNA base excision enzymes and DNA methyltransferases suggests how AGT may
combine different methods of protein-DNA interactions observed in DNA repair
enzymes and DNA methyltransferases.
DNA repair of bulky lesions requires enzymes such as hexameric DNA helicases.
These helicases are amazing molecular motors that unwind DNA for replication,
recombination, and repair. Our diffraction studies on the helicase RuvB will
characterize one of the key helicases involved in recombination repair.
We are also studying another protein key to eukaryotic genome integrity, the
multifunctional enzyme flap endonuclease (FEN-1). Mutations in FEN-1 result in
DNA duplication defects that occur in human tumors and inherited human diseases.
FEN-1 is a structure-specific nuclease necessary for DNA repair and for processing
the 5´ ends of Okazaki fragments during the synthesis of lagging strands
of DNA. Structural analysis of FEN-1 revealed new motifs for DNA binding and
defined the motif for formation of complexes of FEN-1 with the processivity factor
for DNA polymerase, termed proliferating cell nuclear antigen (Fig. 2).
DNA repair and genome fidelity are tightly controlled by checkpoints in the
cell cycle that link DNA repair and replication. Our structures of proteins in
humans that regulate the cell cycle, proteins essential to progression of the
cycle, suggest mechanisms for regulation that involve a unique conformational
switch that controls kinase recognition. Working with S. Reed, The Scripps Research
Institute, we are examining the interactions between radiation-induced DNA damage
and cell-cycle checkpoints.
Genomic variation of human pathogens creates new threats to health in the
form of emerging infectious diseases. Our research in this area focuses on pili,
fibers that not only allow pathogenic bacteria to crawl across host cells but
also act in taking into the cell large regions of foreign DNA containing new
pathogenicity genes. With funding from the Skaggs Institute, we have broadened
our studies to include pili of Vibrio cholerae and Neisseria meningitidis. In
this research, in collaboration with M. Yeager and R. Milligan, The Scripps Research
Institute, we are bridging the resolution gap between protein structures and
supermolecular assemblies by developing new techniques for combining the results
of electron microscopy and x-ray diffraction.
Reactive Oxygen Signals and Defensive Cytotoxins
Human superoxide dismutases protect DNA and other cellular components from
the oxidative damage caused by superoxide that occurs in degenerative diseases
such as Lou Gehrig disease, cancer, and aging. As part of our work on these enzymes,
we solved structures of human catalase, an enzyme that protects cells against
oxidative damage and cell death caused by hydrogen peroxide. These structures
help define the catalase mechanism, suggest a role for the cofactor NADPH, and
establish the basis for activity of an important heterocycle inhibitor.
A second major focus for our research on the regulation of reactive oxygen
is structural studies of nitric oxide synthases (NOSs), done in collaboration
with E. Getzoff, the Skaggs Institute. These enzymes oxidize arginine to make
nitric oxide, a key intercellular signal and defensive cytotoxin in the nervous,
cardiovascular, and immune systems. Overproduction of nitric oxide by inducible
NOS (iNOS) in macrophages can lead to many abnormalities, including juvenile
diabetes, arthritis, aneurysms, neurodegenerative disorders, and septic shock.
Consequently, specific inhibitors of iNOS are of great therapeutic potential.
The structures of ligands bound to NOS suggest new aspects of the enzyme mechanism
and aid in the design of specific inhibitors.
Guan, Y., Manuel, R.C., Arvai, A.S., Parikh, S.S., Mol, C.D., Miller, J.H.,
Lloyd, S., Tainer, J.A. MutY catalytic core, mutant and bound adenine structures
define specificity for DNA repair enzyme superfamily. Nat. Struct. Biol. 5:1058,
Hosfield, D.J., Frank, G., Weng, Y., Tainer, J.A., Shen, B. Newly discovered
archaebacterial flap endonucleases show a structure-specific mechanism for DNA
substrate binding and catalysis resembling human flap endonuclease-1. J. Biol.
Chem. 273:27154, 1998.
Hosfield, D.J., Mol, C.D., Shen, B., Tainer, J.A. Structure of the
DNA repair and replication endonuclease and exonuclease FEN-1: Coupling DNA and
PCNA binding to FEN-1 activity. Cell 95:135, 1998.
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 Dismutases: Recent
Advances and Clinical Applications. Edeas, M.A. (Ed.). Editions Mel Paris, Paris,
France, 1999, p. 22.
Mol, C.D., Parikh, S.S., Lo, T.P., Tainer, J.A. Structural phylogenetics
of DNA base excision repair. Nucleic Acids Mol. Biol. 12:29, 1998.
Parikh, S.S., Mol, C.D., Hosfield, D.J., Tainer, J.A. Envisioning the
molecular choreography of DNA base excision repair. Curr. Opin. Struct. Biol.
Parikh, S.S., Mol, C.D., Slupphaug, G., Bharati, B., Krokan H.E., Tainer,
J.A. Base excision repair initiation revealed by crystal structures and binding
kinetics of human uracil-DNA glycosylase with DNA. EMBO J. 17:5214, 1998.
Putnam, C.D., Shroyer, M.J.N., Lundquist, A.J., Mol, C.D., Arvai, A.S.,
Mosbaugh, D.W., Tainer, J.A. Protein mimicry of DNA from crystal structures
of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia
coli uracil-DNA glycosylase. J. Mol. Biol. 287:331, 1999.
Shen, B., Qiu, J., Hosfield, D., Tainer, J.A. Flap endonuclease homologs
in archaebacteria exist as independent proteins. Trends Biochem. Sci. 25:171,