1. 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 their 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 Pneumocystis 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 structural study, homodimers of E. coli DHFR in complex with bifunctional inhibitors are being carried out to develop new methods of drug delivery.

2. 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 10⁵-fold.  Diffraction data were measured for the wild type and H101A mutant 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. 

3. E. coli Beta Sliding Clamp

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.  We previously described a mutant form of the E. coli beta sliding clamp protein bearing alanine substitutions in place of residues 148-152 (beta-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 beta-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 beta-148-152 clamp protein. These data reveal that the loop encompassing the mutations adopts an alternative conformation from the wild type protein.   These data suggest that these residues play a vital role in DNA polymerase switching.

4. 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. 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.  To model potential interactions of thyroid and steroid hormone analogues with integrin, molecular docking combined with quantum chemical calculations were carried out.  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. 

5. Drosophila GTP-Cyclohydrolase I

Catastrophic loss of dopaminergic neurons is a hallmark of Parkinson’s disease.  Environmental toxins such as the herbicide paraquat have been shown to reduce the number of dopaminergic neurons, and to be associated with increased incidence of Parkinson’s disease in some populations.  Biopterin (BH4), the regulating cofactor of tyrosine hydroxylase which catalyzes the first and rate-limiting step in dopamine biosynthesis pathway, is itself synthesized from GTP (guanosine triphosphate) by three enzymatic reactions of which GTP CH-I is the rate limiting step.  GTP CH-I catalyses the formation of dihydroneopterin triphosphate from GTP and is the first committed intermediate in the biosynthetic pathway of tetrahydrofolate in plants and microorganisms, and of tetrahydrobiopterin in animals.  It has been shown that GTP CH-I gene mutations cause biopterin deficiency that impairs dopamine synthesis.  The Drosophila system has been shown an effective model of the effects of environmental toxin-induced parkinsonism as it is a genetically tractable organism to model gene-environment nteractions that could be a beneficial and useful means of identifying genetic risk factors.

Structural studies are underway to understand how the Drosophila GTP cyclohydrolase differs from the human protein and to understand the mechanism of action

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