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Mutational analysis of CCL20 reveals flexibility of N-terminal amino acid composition and length. J Leukoc Biol 2018 08;104(2):423-434

Date

08/17/2018

Pubmed ID

30114340

DOI

10.1002/JLB.1VMA0218-049R

Scopus ID

2-s2.0-85050596102   1 Citation

Abstract

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 (NH -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.

Author List

Riutta SJ, Larsen O, Getschman AE, Rosenkilde MM, Hwang ST, Volkman BF

Author

Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Chemokine CCL20
DNA Mutational Analysis
Humans
Jurkat Cells
Protein Binding
Receptors, CCR6
Recombinant Proteins
jenkins-FCD Prod-444 eb4ebd1a08581aba961d3befd3b851a3c3ec6b46