Research Summary Protein Structure Our laboratory is investigating the structural basis of intercellular signaling via chemotaxis and the role of chemokines in the process of viral infection, as well as correlating the structures of defensins with their antimicrobial and chemotactic properties. We are also studying the enzymatic mechanism and specificity of L-asparaginases. Although we primarily use x-ray crystallography in our studies, our research techniques also extend into biochemistry and molecular biology. Our recent efforts have been concentrated in three distinct areas: (1) chemokines and chemokine receptors, (2) defensins, and (3) L-asparaginases. Chemokines and Chemokine Receptors Chemokines have been recognized for many years as proteins associated with inflammation, making them interesting targets for the treatment of immune disorders. Recent discoveries link chemokines and chemokine receptors to processes such as viral infection, hematopoiesis, and cancer metastasis, thus significantly broadening the biological role of this system and creating the potential for their use as therapeutic targets. We are particularly interested in understanding the structural factors that determine the interactions between chemokines and their receptors. Despite the extensive overall structural similarity among all chemokines, individual proteins display a significant degree of specificity toward their receptors. We believe this specificity can be understood by analyzing the structural features of specific chemokines. The x-ray structures of MCP-1, fractalkine, Met-, and AOP-RANTES, studied previously by our laboratory, have allowed us to correlate some important properties of these proteins with the topological features of their molecules. However, more general correlations will require a significantly larger amount of structural data; we are currently acquiring this information through our studies of novel members of the chemokine family and their mutants and/or analogs. Another, more challenging, component of this project will aim at understanding the structural features of chemokine receptors. Defensins Defensins are small basic proteins that, until recently, were mainly known for their potent antimicrobial properties. We now know that human beta-defensins are also potent chemoattractants, and that they specifically interact with CCR6—the receptor for the chemokine MIP-3alpha. The mechanisms underlying the antimicrobial and chemotactic activity of defensins is not well understood, although both of these properties are of practical interest. The crystal structures of human beta-defensin-2, solved by our laboratory, were the first published for human beta-defensins. These structures revealed a topology and an oligomerization mode for defensins that had not been reported previously. Our findings shed new light on a possible mechanism for the antimicrobial properties of these proteins, and we recently extended our research by solving the x-ray structure of another human beta-defensin, hBD1. One of our primary goals in studying defensins is to establish the molecular basis of their chemotactic properties, which greatly complements our interest in chemokines. Additionally, we will focus on determining the mechanism of the antimicrobial activity of these proteins. Future studies will involve numerous mutant proteins and will be complemented by extensive biological assays. L-Asparaginases L-asparaginases, in particular two bacterial enzymes from Escherichia coli and Erwinia chrysanthemi, have been used for nearly 30 years to treat certain leukemias and lymphomas. However, the moderately low specificity and immunological incompatibility of bacterial L-asparaginases results in the severe side effects observed during therapeutic applications involving these enzymes. Our longstanding interest in bacterial L-asparaginases focuses on understanding their enzymatic activity and substrate specificity. Thus far, we have determined the structures of L-asparaginases from five bacterial sources, including their mutants, as well as the complexes they form with substrates and inhibitors. Our results have allowed us to correlate, in detail, the structural characteristics of specific enzymes with their unique substrate specificities. We have formulated a model for the mechanism of catalysis by L-asparaginases that agrees with all of the structural and kinetic information available. At present, our efforts are directed toward the cloning, purification, and crystallization of human L-asparaginase, as the genomic sequence for this enzyme is now available.