Receptor-binding conformation of the "ELR" motif of IL-8: X-ray structure of the L5C/H33C variant at 2.35 A resolution.

The "ELR" (Glu-Leu-Arg) tripeptide sequence near the N-terminus of interleukin-8 (IL-8) contributes a large part of the receptor binding free energy. Prior X-ray and nuclear magnetic resonance (NMR) structures of IL-8 have shown this region of the molecule to be highly mobile. We reasoned that a hydrophobic interaction between the leucine and the neighboring beta-turn might exist in the receptor binding conformation of the N-terminus. To test this hypothesis, we mutated two residues to cysteine and connected the N-terminus to the beta-turn. The mutant retains receptor binding affinity reasonably close to wild type and allows the characterization of a high-affinity conformation that may be useful in the design of small IL-8 mimics. The L5C/H33C mutant is refined to R-values of R = 20.6% and Rfree = 27.7% at 2.35 A resolution. Other receptor binding determinants reside in the "N-loop" found after "ELR" and preceding the first beta-strand. All available structures of IL-8 have been found with one of two distinct N-loop conformations. One of these is relevant for receptor binding, based on NMR results with receptor peptides. The other conformation obscures the receptor-peptide binding surface and may have an undetermined but necessarily different function.