Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1 alpha) and a co-receptor for the entry o f specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflamm atory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1 al pha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-recepto r. An approach combining protein structural modeling and site-directed muta genesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1 alpha and vMIP-II and HIV-1 envelope p rotein gp120. Hypothetical three-dimensional structures were proposed by mo lecular modeling studies of the CXCR4-SDF-1 alpha complex, which rationaliz e extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N -terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellula r loop 2 (ECL2) impairs the signaling mediated by SDF-1 alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 a re involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of nove l targeted inhibitors.