Mutational analysis of CCL20 reveals flexibility of N-terminal amino acid composition and length
| Autor: | Olav Larsen, Samuel T Hwang, Anthony E. Getschman, Sarah J. Riutta, Brian F. Volkman, Mette M. Rosenkilde |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Receptors CCR6 Chemokine Immunology DNA Mutational Analysis chemical and pharmacologic phenomena C-C chemokine receptor type 6 Biology medicine.disease_cause 03 medical and health sciences Jurkat Cells 0302 clinical medicine medicine Immunology and Allergy Humans Amino Acid Sequence Receptor Alanine Mutation Chemokine CCL20 hemic and immune systems Chemotaxis Cell Biology Ligand (biochemistry) Recombinant Proteins Cell biology CCL20 030104 developmental biology 030220 oncology & carcinogenesis biology.protein Protein Binding |
| Zdroj: | Journal of leukocyte biology. 104(2) |
| ISSN: | 1938-3673 |
| Popis: | Chemokine–chemokine receptor (CKR) interactions are traditionally described by a two-step/two-site mechanism that details the major contact points between chemokine ligands and CKRs leading to ligand recognition and receptor activation. Chemokine recognition site 1 (CRS1) encompasses interactions between the CKR N-terminus and the globular chemokine core. Chemokine recognition site 2 (CRS2) includes interactions between the unstructured chemokine N-terminus and the binding pocket of the receptor. The two-step/two-site paradigm has been an adequate framework to study the intricacies of chemokine:CKR interactions, but emerging studies highlight the limitations of this model. Here, we present studies of CRS2 interactions between the chemokine CCL20 and its cognate receptor CCR6 driven by the hypothesis that CCL20 interacts with CCR6 as described by the two-step/two-site model. CCL20 is a chemokine with an unusually short N-terminus of 5 residues (NH2-ASNFD), compared to the average length of 10 residues for chemokine ligands. We have investigated how well CCL20 tolerates manipulation of the N-terminus by monitoring binding affinity of variants and their ability to activate the receptor. We show the CCL20 N-terminus tolerates truncation of up to 3 residues, extension by up to 5 additional residues, and point mutations at 4 of 5 positions with minimal loss of binding affinity and minimal impairment in ability to stimulate calcium mobilization, inositol triphosphate accumulation, chemotaxis, and β-arrestin-2 recruitment. Mutation of the fifth residue, aspartate, to alanine or lysine has a dramatic impact on binding affinity for CCR6 and ligand potency. We postulate CCL20 does not activate CCR6 through the canonical two-step/two-site mechanism of CKR activation. Systematic structure-function analysis of the CCL20 N-terminus reveals extreme tolerance of N-terminal extension, truncation, or modification to maintain activity at CCR6. |
| Databáze: | OpenAIRE |
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