CC Chemokine MIP-1β Can Function As a Monomer and Depends on Phe13 for Receptor Binding

Abstract

The reported structures of many CC chemokines show a conserved dimer interface along their N-terminal region, raising the possibility that the quaternary arrangement of these small immune proteins might influence their function. We have produced and analyzed several mutants of MIP-1 beta having a range of dimer K(d) values in order to determine the significance of dimerization in receptor binding and cellular activation. NMR and analytical ultracentrifugation were used to analyze the oligomeric state of the mutants. Functional relevance was determined by receptor binding affinity and the ability to invoke intracellular calcium release from CHO cells transfected with the MIP-1 beta receptor CCR5. The monomeric N-terminally truncated mutant MIP(9) was able to bind the CCR5 receptor with a K(i) of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability to tightly bind (K(i) = 480 pM) and to activate (EC(50) = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP-1 beta, as a key determinant for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic side chain to be important for both binding to CCR5 and chemokine dimerization.

Control of feeding behavior inC. elegansby human G protein-coupled receptors permits screening for agonist-expressing bacteria

“…5). The hydrophobic core includes F12, whose aromatic side chain has a pivotal role in CCR5 binding (6,(12)(13)(14)(15)(16)(17)(18). Indeed, our findings show a dramatic disruption in the ability to bind CCR5, when this residue is changed to alanine, validating the importance of the aromatic side chain for its binding activity.…”

“…The replacement of N-terminal residues, T8 with alanine and A9 with glutamine, resulted in complete loss of activity, indicating the importance of a hydrophilic residue in position 8, and a hydrophobic amino acid in position 9 for receptor activation. The corresponding threonine residue in MIP-1␤ is important in dimer formation given its location at the hydrophobic dimer interface, where mutation to a charged residue will lead to a monomeric form (12). Interestingly, T8A mutation has no effect on the activity of RANTES with CCR1 (15).…”

Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1β

“…The present work displays the structural underpinning of the high affinity between poxvirus vCCI and CC chemokines. In the vCCI:MIP-1␤ structure, vCCI makes no contact with the first seven chemokine residues, which are involved in receptor activation (8). However, the highly conserved vCCI residues Ser-182 to Thr-187 make extensive contacts with the MIP-1␤ residues Pro-8 to Ser-14.…”

“…Unlabeled, 15 N-labeled, 13 C, 14 Nlabeled, and 13 C, 15 N-labeled MIP-1␤ 45 AASA 48 were produced and purified by following the protocol described in ref. 8.…”

“…Despite the differences in amino acid composition and functionalities, most chemokines share a remarkably conserved tertiary structure, with an extended N terminus followed by three ␤-strands in a Greek key arrangement and a C-terminal ␣-helix. The solution structure of the human CC chemokine MIP-1␤ (macrophage inflammatory protein 1␤) revealed a homodimer (7), and, subsequently, the dimer dissociation constant was determined to be 0.73 M (8). Dimerization of other chemokines has been observed under in vitro conditions, and the ability to dimerize is necessary for some chemokines to function in vivo (9).…”