A homology model of GROγ, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GROγ. Residues Tyr 13, Ser 14, Phe 21, and Lys 49 are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Individual amino acid substitutions were made at positions where IL-8 and GROγ sequences differ within the regions of residues 11-21 and 46-53. The amino-terminal loop (amino acids 1-18) and the third β-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. When IL-8 sequences were substituted into GROγ, a single domain containing the second β-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. Exchanges of the second β-sheet (amino acids 32-46) or the carboxyl-terminal α-helix (amino acids 53-72) had no significant effect. The third β-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Substitution into IL-8 of the GROγ sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first β-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GROγ, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity.
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