The chemokine receptors CCR5 and CXCR4, in conjunction with CD4 and the viral envelope glycoprotein (Env), mediate infection by human immunodeficiency virus type 1 (HIV-1). Our long-term objective is to understand the structural features of chemokine receptors that enable them to function as portals for HIV-1 entry. Such structural knowledge will provide valuable insight for designing receptor decoys as therapeutics for HIV-related diseases. The current cocktail treatment has dramatically reduced mortality, but the viruses can mutate and become resistant to the existing drugs that inhibit viral protein function. Therefore, targeting host proteins such as the CCR5 chemokine receptor provides an attractive alternative strategy. A cluster of charged amino acids and sulfated tyrosines in the CCR5 receptor N-terminal domain (N-domain) play a critical role in HIV binding. Tyrosine-sulfated N-domain peptides competitively inhibit HIV binding to the receptor, and facilitate HIV binding to a mutant receptor lacking the N-domain. Our preliminary data show that the receptor N-domains are unstructured in solution, but structured in detergent micelles that mimic the """""""" native membrane environment. These observations suggest that the receptor N-domain functions as an autonomous folded structural and functional unit. This allows studying isolated receptor N-domain to gain insight into the native domain's structure and flexibility, and binding-induced structural and dynamic changes. Nuclear magnetic resonance (NMR) spectroscopy is ideally suited for such studies, but requires large (mg) quantities of recombinant protein, preferably isotopically labeled (13C and 15N) and tyrosine-sulfated.
In aim 1, we will express the CCR5 N-domain both in E.coli and insect cells, and optimize conditions for its high-level production, purification, solubility, and sulfation.
In aim 2, we will use NMR and other biophysical techniques to characterize the structure of the free and ligand-bound CCR5 N-domain. We will use an in vitro assay to test the receptor domain peptides ability to inhibit HIV infection. Our approaches are novel, and successful accomplishment of our aims is critical for understanding the structural role of CCR5 in HIV infection. Most importantly, receptor decoys designed on the basis of CCR5 N-domain structure should result in drugs for preventing/treating HIV-related diseases.
|Haldar, Sourav; Raghuraman, H; Namani, Trishool et al. (2010) Membrane interaction of the N-terminal domain of chemokine receptor CXCR1. Biochim Biophys Acta 1798:1056-61|
|Fernando, Harshica; Nagle, Gregg T; Rajarathnam, Krishna (2007) Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain. FEBS J 274:241-51|
|Rajagopalan, Lavanya; Rajarathnam, Krishna (2006) Structural basis of chemokine receptor function--a model for binding affinity and ligand selectivity. Biosci Rep 26:325-39|