CC chemokine MIP-1 beta can function as a monomer and depends on Phe13 for receptor binding.
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.
Laurence
J S
JS
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Blanpain
C
C
Burgner
J W
JW
Parmentier
M
M
LiWang
P J
PJ
D000409:Alanine / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D054407:Chemokine CCL4; D019742:Chemokines, CC / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006224:Cricetinae; D019281:Dimerization; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D019713:Receptors, CCR5 / Q000378:metabolism; D019707:Receptors, Chemokine / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection; D014461:Ultracentrifugation
D054407:Chemokine CCL4; D019742:Chemokines, CC; D019402:Macrophage Inflammatory Proteins; D010446:Peptide Fragments; D019713:Receptors, CCR5; D019707:Receptors, Chemokine; D010649:Phenylalanine; D000409:Alanine