Molecular Recognition in Biological Systems and Bioinformatics

Protein structure and function; protein structure determination; crystallization.

Structure-Activity Correlations for Dihydrofolate Reductase Inhibition

In order to understand the role of key active site residues among fungal, mammalian, and bacterial species of dihydrofolate reductase (DHFR), mutational studies are being carried out to measure the influence on binding specificity and selectivity. Structural studies of inhibitor complexes with various DHFR species (human, mouse, rat, beef, Pneumocystis carinii, Pneumocystis jirovecii and E. coli DHFR) are under investigation. These data have revealed novel modes of binding for potent inhibitors and show that the dynamics of binding can control the preferred conformation for binding.

The current focus is on the design of inhibitors that are selective for the inhibition of DHFR from the opportunistic pathogen Pneunocystis jirovecii, the causative agent for AIDS-related pneumonia. Mutation of key active site residues are being carried out to measure the inhibitory and kinetic effect of inhibitors on selectivity and specificity of DHFR inhibition.

In a second study, homodimers of E. coli DHFR in complex with bifunctional inhibitors is being carried out to develop new methods of drug delivery.

Histidine Triad Nucleotide Binding Proteins (Hint)

The histidine triad nucleotide proteins (Hints) from human and E. coli have been established as purine nucleoside phosphoramidases. Human Hint1 possesses tumor suppressor activity whereas E. coli HinT is involved in high salt tolerance for E. coli. The homodimeric Hint proteins are characterized by a His-X-His-X-His-XX motif where X is a hydrophobic residue. Additionally, human HinT1 and its bacterial homolog have different substrate specificities. To understand the mechanism of substrate specificity, kinetic and structural studies of mutants that replace the active site histidines were carried out. These data showed that H101A decreased specific activity by 10^5-fold. Diffraction data were measured for the wild type (1.5Å resolution) and H101A mutant (1.7Ǻ) ecHinT GMP complexes. There are four unique homodimers in the wild type, and two homodimers in the mutant structure. The overall fold of the ecHinT structures is similar to human HinT1.

E. coli Beta Sliding Clamp (in collaboration with Mark Sutton, Biochemistry)

Damaged bases in the DNA that are not repaired prior to replication can act as potent blocks to polymerization, leading to replication fork arrest. One mechanism by which these lesions are tolerated involves their direct bypass by a specialized DNA polymerase (pol) via a process termed translesion DNA synthesis (TLS). In E. coli, most TLS following UV irradiation is catalyzed by the umuDC-encoded pol V. Sutton has previously described a mutant form of the E. coli sliding clamp protein bearing alanine substitutions in place of residues 148-152 (-148-152) that was severely impaired for pol IV-dependent TLS in vivo. Inactivation of pol II and pol IV fully restored pol-V-dependent UV-induced mutagenesis in the -148-152 mutant in vivo. As part of an effort to understand the mechanistic basis for this phenotype, we have solved the crystal structure of the mutant -148-152 clamp protein. Data were measured to 1.75Å resolution for the homodimeric E. coli -148-152 mutant that crystallizes in a triclinic lattice. These data reveal that the loop encompassing the mutations adopts an alternative conformation from the wild type protein. In addition, BIAcore and gel filtration chromatography data for the interactions of wild type and -148-152 clamp proteins with various E. coli pols indicate that residues 148-152 define a surface of the clamp that is critically important for the proper functioning of some, but not all, E. coli pols. These data suggest that these residues play a vital role in DNA polymerase switching.

Thyroid Hormone Integrin Interactions

Integrins are ubiquitous heterodimeric structural proteins of the cell membrane that convey signals to and from the cell interior to the extracellular matrix. A novel cell surface receptor for thyroid hormone has been identified on the extracellular domain of integrin alphaVbeta3 that leads in a variety of human cell lines to activation by the hormone of the mitogen-activated protein kinase (MAPK) signal transduction cascade within the cell. The arginine-glycine-aspartate (RGD) recognition site on integrin is essential to the binding of a variety of extra cellular matrix proteins. Recent competition data reveal that RGD peptides block hormone-binding (T4ac inhibits T4-induced MAPK activity) suggesting that the hormone interaction site is located near the RGD recognition site on integrin alphaVbeta3. Structural data show that an RGD cyclic peptide binds at the interface of the propeller of the alphaV and the B domains on the integrin head (Arnaout, et. al, Science, 2002). To model potential interactions of thyroid (T4, T4ac, T3, GC-1) and steroid (E2, resveratrol, stilene) hormone analogues with integrin, molecular docking combined with quantum chemical calculations were carried out in collaboration with Furlani and Freindorf, CCR. These results are compared to previous modeling studies (Cody, et al, Steroids, 2007) that indicated the thyroid hormones had preferential binding at the surface of the B domain whereas the more planar molecules could bind in an alternate pocket. These computational results showed preferential binding to the alternate site and showed there was a strong electronic contribution to binding energies by the presence of Mg near the active site that impacts ligand binding. These data show that CG-1, T4ac and c-resveratrol have the strongest stabilization energies of the compounds tested. These calculations also showed that there can be significant difference in the binding orientation of similar liagands.

X-ray diffraction

Dihydrofolate reductase, beta sliding clamp, histidine triade nucleotide binding protein, alpha V beta 3 integrin

Treatment of AIDS-related pneumonia, cancer, thyroid disease

Cloning, expression, purification, crystallization

AIDS pneumonia, cancer, thyroid disease

E. coli, baculovirus

http://www.hwi.buffalo.edu/Faculty/Cody