Chemokines are small soluble proteins of about 70 amino acid residues with a molecular mass of 8 to 10 kDa. They play prominent roles in leukocyte activation and trafficking to sites of inflammation by interacting with chemokine receptors. All known human chemokines are categorized based on the positions of two conserved cysteine residues in their amino (N)-terminal domains. The two major classes are the CXC and CC chemokines (1
). The chemokine receptor CXCR4, a member of the superfamily of G-protein-coupled receptors (GPCRs) possessing seven transmembrane (TM) helical domains, specifically binds the CXC chemokine stromal cell-derived factor 1α (SDF-1α), triggering multiple intracellular signals (1
). Human immunodeficiency virus type 1 (HIV-1) requires a coreceptor, either CXCR4 or CCR5, for entry into target cells in addition to CD4, the primary receptor on the target cell surface. CXCR4 is the principal coreceptor for the T-cell-line-tropic HIV-1 isolate which is involved in the onset of AIDS-defining symptoms (1
The HIV-1 envelope (Env) consists of gp120 and gp41. While gp120 contains the CD4 binding site, gp41 contains a hydrophobic fusion peptide directly involved in membrane fusion. In a plausible model, CD4 binding induces conformational changes in gp120 that expose the coreceptor binding determinants. The gp120 interaction with the coreceptor then induces a further conformational change in Env that results in insertion of the fusion peptide into the target cell membrane (1
). This suggests an important role for CXCR4 as a potential target to combat the AIDS epidemic. Small-molecule inhibitors of CXCR4 have been described (8
). However, there is cause for concern regarding undesired side effects of blocking the normal CXCR4-SDF-1α function, since knockout mice lacking either CXCR4 (28
) or SDF-1α (22
) die during embryogenesis, with evidence of hematopoietic, cardiac, vascular, and cerebellar defects. As a result, it would be desirable to develop compounds that can target specific regions of CXCR4 that are selective for HIV-1 coreceptor function only and not for the normal function of SDF-1α.
To develop such selective inhibitors of CXCR4, it is essential to first understand the mechanisms by which CXCR4 interacts with HIV-1 gp120 versus the normal ligand SDF-1α. Previous structure-function studies of CXCR4 have shown that there is significant overlap between HIV-1 and chemokine functional sites on the extracellular regions of CXCR4 (4
). However, it has yet to be determined whether residues and sites located near or within the TM helices of CXCR4 might play any structural and functional roles in ligand interactions, and if they do, whether such roles are different for different ligands (i.e., HIV-1 gp120 and SDF-1α). To address these questions, we constructed a panel of mutations at residues near or within the TM helices based on the following considerations: (i) charged residues such as D97, D171, and E288 might be involved in interactions with the positively charged residues of SDF-1α; (ii) residues such as H79, Y121, W161, Y219, N298, and Y302 are either highly conserved among chemokine receptors or analogous to corresponding sites in other GPCRs found to be functionally important (27
); and (iii) residues such as P163 might play a role in affecting the helical conformation of CXCR4. In addition to these TM mutants, several mutants of the second extracellular loop (ECL2) residues were constructed to investigate the role of ECL2 in HIV-1 entry versus natural ligand binding and signaling. Through these studies, we hope to identify potentially different determinants for CXCR4 interactions with HIV-1 gp120 and SDF-1α and eventually use such information for the design of novel inhibitory molecules specifically targeting the CXCR4-HIV-1 gp120 interaction only.