Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.
Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.
Navenot
J M
JM
Brown Cancer Center, University of Louisville, Louisville, KY, USA.
Wang
Z X
ZX
Trent
J O
JO
Murray
J L
JL
Hu
Q X
QX
DeLeeuw
L
L
Moore
P S
PS
Chang
Y
Y
Peiper
S C
SC
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001667:Binding, Competitive; D065100:CCR5 Receptor Antagonists; D016466:CHO Cells; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018925:Chemokines / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D006023:Glycoproteins / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D019402:Macrophage Inflammatory Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D065100:CCR5 Receptor Antagonists; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5; D018925:Chemokines; D006023:Glycoproteins; D008024:Ligands; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C108059:vMIP-II; D000409:Alanine