Chemokines (chemotactic cytokines) comprise a large family of proteins that recruit and activate leukocytes, giving chemokines a major role in both the immune response and inflammation-related diseases. Accordingly, there is a great deal of interest in understanding and modulating the action of these proteins. The poxvirus-encoded viral chemokine inhibitor (vCCI) is a 26 kDa protein that is able to bind many members of the CC chemokine subfamily with high affinity, acting as a potent anti-inflammatory agent by abrogating CC chemokine activity. The long-range goals of the research are to understand chemokine binding strategies at a molecular level, and to eventually postulate strategies to inhibit inflammatory diseases such as asthma and rheumatoid arthritis. In this proposal we seek to understand the structural basis for the specificity and affinity of the binding of vCCI with members of the CC chemokine subfamily.
In Specific Aim 1, heteronuclear NMR (nuclear magnetic resonance) will be used to determine the structure of the complex between vCCI and the CC chemokine MIP-1 p.
In Specific Aim 2 the study will be extended to include complexes between vCCI and other CC chemokines to allow both NMR and Xray crystallographic analysis of how vCCI is able to bind multiple partners with subnanomolar affinity.
In Specific Aim 3 structure-based predictions of the key intermolecular vCCI-chemokine interactions will be tested using biophysical measurements on mutant proteins. First, minimal mutations will be designed to abrogate the ability of vCCI to bind chemokines while maintaining folded protein. Later mutations will have the goal of tailoring the specificity of vCCI to optimize this protein's ability to inhibit inflammation, particularly inflammation associated with allergic asthma. The impact of the proposed work is that an understanding of strategies used by vCCI to circumvent the chemokine response may lead to new insights for targeted therapeutics. Lay language relevance: Chemokines are pro-inflammatory proteins that are involved in a wide range of diseases, including asthma, atherosclerosis, and rheumatoid arthritis. A structural picture of how vCCI binds and inhibits chemokines may allow the adoption of similar strategies in new medicines to treat these diseases. ? ? ?
