High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Coreceptor CCR5.
- Rico CA Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York; Tri-Institutional PhD Program in Chemical Biology, The Rockefeller University, New York, New York.
- Berchiche YA Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York; B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland.
- Horioka M Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York; Tri-Institutional PhD Program in Chemical Biology, The Rockefeller University, New York, New York.
- Peeler JC Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York; Deparment of Chemistry, Boston College, Chestnut Hill, Massachusetts.
- Lorenzen E Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York.
- Tian H Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York.
- Kazmi MA Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York.
- Fürstenberg A Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York; Department of Inorganic and Analytical Chemistry, Geneva, Switzerland.
- Gaertner H Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland.
- Hartley O Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland.
- Sakmar TP Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York.
- Huber T Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York. Electronic address: hubert@rockefeller.edu.
- 2019-08-19
Published in:
- Biophysical journal. - 2019
Binding, Competitive
Chemokine CCL5
Chemokines
HEK293 Cells
HIV-1
Humans
Ligands
Models, Biological
Protein Binding
Receptors, CCR5
English
The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.
- Language
-
- English
- Open access status
- bronze
- Identifiers
-
- DOI 10.1016/j.bpj.2019.07.043
- PMID 31421836
- Persistent URL
- https://folia.unifr.ch/global/documents/46547
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