Structural and Functional Characterization of Human CXCR4 as a Chemokine Receptor and HIV-1 Co-receptor by Mutagenesis and Molecular Modeling Studies

Abstract

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1␣) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1␣ or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1␣ and vMIP-II and HIV-1 envelope protein gp120. Hypothetical threedimensional structures were proposed by molecular modeling studies of the CXCR4⅐SDF-1␣ complex, which rationalize 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 extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1␣. As an HIV-1 co-receptor, we found that the Nterminal domain, ECL2, and ECL3 of CXCR4 are 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 novel targeted inhibitors.Chemokines are a family of 8 -10-kDa small proteins that act as chemoattractants of various types of leukocytes to sites of inflammation and to secondary lymphoid organs. Based on the positions of two conserved cysteine residues in their N termini, chemokines can be divided into four subfamilies: CC, CXC, CX3C, and C (1, 2). The stromal cell-derived factor-1 (SDF-1␣) 1 is one of the CXC chemokines, which plays critical roles in the migration, proliferation, and differentiation of leukocytes. SDF-1␣ is the only known natural ligand of CXCR4 receptor (3, 4). CXCR4 can also be recognized by an antagonistic ligand, the viral macrophage inflammatory protein-II (vMIP-II) encoded by the Kaposi's sarcoma-associated herpesvirus (5). vMIP-II acts as a selective chemoattractant for T helper 2 cells and monocytes and is an agonist for CCR8 (6). vMIP-II displays a broader spectrum of receptor activities than any mammalian chemokine, as it binds with high affinity to a number of both CC and CXC chemokine receptors including CXCR4 and CCR5 and inhibits cell entry of human immunodeficiency virus type I (HIV-1) mediated by these receptors (7,8).CXCR4 belongs to the family of seven transmembrane Gprotein-coupled receptors that transduce signals via heterotrimeric G-proteins (9). Recent studies with knockout mice of CXCR4 have demonstrated that this molecule plays an important role in immunomodulation, organogenesis, hematopoiesis, and derailed cerebellar neuron migration (10 -12). CXCR4 has...

Probing the Molecular Interactions between CXC Chemokine Receptor 4 (CXCR4) and an Arginine-Based Tripeptidomimetic Antagonist (KRH-1636)

“…In line with the findings in the present study, the D97A, D187A, and E288A mutations have previously been found to affect both the binding and signaling of CXCL12. 37,[45][46][47][48][49] Moreover, Trp 94 and Tyr 116 , together with the three aforementioned residues, have been implicated as part of "site two" in the "two-site model" for binding of CXCL12 to CXCR4. 33,45,50,51 Additionally, the direct interaction of Tyr 116 with agonists has been suggested in activation of GPCRs.…”

Lipid Bilayer Simulations of CXCR4 with Inverse Agonists and Weak Partial Agonists

“…Dodecylmaltoside was selected for the lipid bilayer, as it has been used to solubilize biologically active CXCR4 (18). Two previous CXCR4 modeling experiments performed in vacuo (19) and solvated in water (20) …”

Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4

“…At 24 h after viral infection, cells (2-8 Â 10 5 ) were lysed in reporter lysis buffer and assayed for luciferase activity, as per the manufacturer's instructions (Pharmingen), in a luminometer. 31 …”

“…25,26 CXCR4 belongs to the family of seven transmembrane G-proteincoupled receptors. [27][28][29][30][31] These coreceptors transduce signals via heterotrimeric G-proteins. CXCR4 has been clearly identified as one of the major coreceptors for HIV-1 cell entry in vivo.…”

“…One of the problems in designing antichemokine coreceptor therapeutic agents is the complexity of the chemokine receptor-ligand interactions. 31 This represents blocking of a tricomplex, gp120-CD4-CXCR4. The natural ligand of CXCR4 has been identified as stromal cell-derived cofactor (SDF-1), an alpha chemokine.…”