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Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition.

ABSTRACT: C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between the two inhibitors--AK530 had 10-fold-greater CCR5-binding affinity (K(d)=1.4 nM) than AK317 (16.7 nM)-their antiviral potencies were virtually identical (IC(50)=2.1 nM and 1.5 nM, respectively). Molecular dynamics simulations for unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. Indeed, AK530 and AK317, when bound to CCR5, disrupted these interhelix hydrogen bond interactions, a salient molecular mechanism enabling allosteric inhibition. Mutagenesis and structural analysis showed that ECL2 consists of a part of the hydrophobic cavity for both inhibitors, although AK317 is more tightly engaged with ECL2 than AK530, explaining their similar anti-HIV-1 potencies despite the difference in K(d) values. We also found that amino acid residues in the beta-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and binding of the spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits HIV-1 from utilizing the "inhibitor-bound" CCR5 for cellular entry--a mechanism of HIV-1's resistance to CCR5 inhibitors. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants.

SUBMITTER: Maeda K

PROVIDER: S-EPMC2630503 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition.

Maeda Kenji K Das Debananda D Yin Philip D PD Tsuchiya Kiyoto K Ogata-Aoki Hiromi H Nakata Hirotomo H Norman Rachael B RB Hackney Lauren A LA Takaoka Yoshikazu Y Mitsuya Hiroaki H

Journal of molecular biology 20080620 4

C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between th ...[more]

PMID: 18590744

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Project description:The A2a adenosine receptor, a member of the G protein-coupled receptor family, is important in the regulation of dopaminergic pathways of the brain and in platelet and cardiovascular functions. In this study, the role of extracellular loops in ligand binding to the human A2a receptor was explored through site-directed mutagenesis. Four glutamate/aspartate residues (Glu151, Glu161, Glu169, and Asp170) in the second extracellular loop (E2) and a cysteine residue (Cys262) in the third extracellular loop (E3) were individually replaced with alanine and other amino acids. A proline residue (Pro173) in E2 was mutated to arginine, the homologous amino acid in A3 receptors. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. The mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, thus allowing their detection in the plasma membrane with immunological techniques. High affinity specific binding of [3H]2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5'-N-ethylcarboxamidoad eno sine (15 nM) and [3H]8-[4-[[[[2-aminoethyl)-amino]carbonyl]methyl]oxy]phenyl]-1,3- dipropylxanthine (4nM), an A2a agonist and antagonist, respectively, was not observed with four of the mutant receptors, E151A, E151Q, E151D, and E169A, although they were well expressed at the cell surface. The E151A and E169A mutant receptors showed nearly full stimulation of adenylyl cyclase at approximately 10(3)-fold higher concentrations of 2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5'-N-ethylcarboxamidoadenosine . The E161A mutant receptor showed as increase in affinity for the nonxanthine adenosine antagonist 9-chloro-2-(furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine(6 fold) but not for other ligands. An E169Q mutant gained affinity (5-22 fold) for adenosine derivatives (agonists) substituted at N6 but not at C2 or C5' positions. Mutant receptors D170K and P173R were similar to wild-type receptors in binding of both agonist and antagonist radioligands. A C262G mutant also resembled the wild-type receptor in radioligand binding, indicating that a potential disulfide bridge with another cysteine residue in proximity is not required for the structural integrity of the receptor. Our data suggest that certain amino acids in the second extracellular loop may be directly or indirectly involved in ligand binding.

Dataset Information

Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition.

Publications

Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition.

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