We are studying the relationship between protein structure and function, using the technique of high-resolution X-ray diffraction. In the past year, our work has been concentrated in five distinct areas. Enzymes with Anticancer Properties We have been investigating the crystal structures of several members of the family of L-asparaginases, some of which are used clinically as drugs directed against childhood lymphoblastic leukemia. While the mechanism of anticancer activity of these enzymes is not yet clear, we have concentrated on the studies of their enzymatic properties. We have also studied these enzymes complexed with ligands such as glutamate and succinic acid, and we determined the structure of a related protein from Wolinella succinogenes. Another enzyme with potential therapeutic properties is Onconase, a cytotoxic ribonuclease isolated from frog eggs. We have been involved in reengineering this enzyme in order to make it applicable to human cancer therapy and to restore its activity in the absence of posttranslational modifications. Crystal structure of the modified enzyme has been solved. We also solved at atomic resolution the crystal structure of eosinofil-derived neurotoxin (EDN), a related enzyme now being developed as a potential anti-cancer agent. Cytokines and Cytokine Receptors Our section has been investigating the crystal structures of several cytokines and has made progress in preparing their receptor complexes. We have established that a helical cytokine, interleukin-10 (IL-10), is a domain-swapped dimer in which each compact half is composed of fragments of two identical molecules. The structure of a related cytokine encoded in the genome of Epstein-Barr virus has now been determined, providing the first glimpse of the molecular architecture of an agent used by the virus to control the host's immune system. We have solved the crystal structure of IL-19, a novel chemokine related to IL-10, and are attempting to crystallize its complexes with specific receptors. Retroviral Enzymes Enzymes encoded by retroviruses such as HIV are prime targets for designing effective drug therapies. We have been studying the structure of native and drug-resistant HIV-1 protease (PR) complexed with inhibitors, with the aim of tracing the molecular basis of the resistance phenomenon. We have also determined the structures of related enzymes from feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV). The latter PRs are poorly inhibited by most inhibitors of HIV-1 PR, including those in clinical use, although they are capable of cleaving HIV-1-derived sequences. To study the mechanism of drug resistance, we solved the structures of HIV-1, FIV, and EIAV PRs complexed with an identical inhibitor, while the studies of an inactive mutant of FIV PR with a substrate helped in delineating the catalytic mechanism. Another retroviral enzyme under investigation in our laboratory is integrase. We have solved the structure of the catalytic domain of avian sarcoma virus integrase in the presence and absence of divalent cations to atomic resolution, and are attempting cocrystallization of complexes with different substrates. Proteins with antiviral properties We solved the crystal structure of cyanovirin-N, a protein that prevents binding of HIV to its cellular receptor. Since this is accomplished through interactions with high-mannose sugars, we also solved the structure of two such specific complexes, elucidating the mode of binding. We also obtained crystals of another protein with similar properties, scytovirin. Proteolytic enzymes Our laboratory has been working on crystal structures of a number of proteolytic enzymes. We discovered that sedolisin is a member of the subtilisin family, although it is much larger and contains a Ser-Glu-His catalytic triad. We have recently solved the structure of the proteolytic domain of and ATP-dependent E. coli protease Lon, establishing a new fold and a catalytic dyad Ser-Lys for this very interesting enzyme. We have also solved the structure of the alpha domain of Lon.
Showing the most recent 10 out of 52 publications
