Sulfopeptide Probes of the CXCR4/CXCL12 Interface
Reveal Oligomer-Specific Contacts and Chemokine Allostery
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Abstract
Tyrosine
sulfation is a post-translational modification that enhances
protein–protein interactions and may identify druggable sites
in the extracellular space. The G protein-coupled receptor CXCR4 is
a prototypical example with three potential sulfation sites at positions
7, 12, and 21. Each receptor sulfotyrosine participates in specific
contacts with its chemokine ligand in the structure of a soluble,
dimeric CXCL12:CXCR4(1–38) complex, but their relative importance
for CXCR4 binding and activation by the monomeric chemokine remains
undefined. NMR titrations with short sulfopeptides showed that the
tyrosine motifs of CXCR4 varied widely in their contributions to CXCL12
binding affinity and site specificity. Whereas the Tyr21 sulfopeptide
bound the same site as in previously solved structures, the Tyr7 and
Tyr12 sulfopeptides interacted nonspecifically. Surprisingly, the
unsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12
monomer that is inaccessible in the CXCL12 dimer. Functional analysis
of CXCR4 mutants validated the relative importance of individual CXCR4
sulfotyrosine modifications (Tyr21 > Tyr12 > Tyr7) for CXCL12
binding
and receptor activation. Biophysical measurements also revealed a
cooperative relationship between sulfopeptide binding at the Tyr21
site and CXCL12 dimerization, the first example of allosteric behavior
in a chemokine. Future ligands that occupy the sTyr21 recognition
site may act as both competitive inhibitors of receptor binding and
allosteric modulators of chemokine function. Together, our data suggests
that sulfation does not ubiquitously enhance complex affinity and
that distinct patterns of tyrosine sulfation could encode oligomer
selectivity, implying another layer of regulation for chemokine signaling