Multiple charged and aromatic residues in CCR5 amino-terminal domain are involved in high affinity binding of both chemokines and HIV-1 Env protein.
CCR5 is a functional receptor for MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major determinant for chemokine binding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residues in the CCR5 amino-terminal domain for chemokine binding, functional response to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the binding affinity for chemokines, which correlated with a similar drop in functional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak responses to high concentrations (500 nM) of RANTES or MIP-1beta. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env proteins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alanine-scanning mutagenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18) that played an important role in both chemokine and Env high affinity binding. The overlapping binding site of chemokines and gp120 on the CCR5 amino terminus, as well as the involvement of these residues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.
Blanpain
C
C
IRIBHN, Université Libre de Bruxelles, Campus Erasme, 808 Route de Lennik, B-1070 Bruxelles, Belgium.
Doranz
B J
BJ
Vakili
J
J
Rucker
J
J
Govaerts
C
C
Baik
S S
SS
Lorthioir
O
O
Migeotte
I
I
Libert
F
F
Baleux
F
F
Vassart
G
G
Doms
R W
RW
Parmentier
M
M
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D018925:Chemokines / Q000378:metabolism*; D006224:Cricetinae; D005434:Flow Cytometry; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D019713:Receptors, CCR5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018925:Chemokines; D015699:HIV Envelope Protein gp120; D019713:Receptors, CCR5; D000409:Alanine