Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket.
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 GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop (amino acids 1-18) and the third beta-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. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues Tyr13, Ser14, Phe21, and Lys49 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 homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma. These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding. Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity.
Hammond
M E
ME
Chiron Corporation, Emeryville, California 94608, USA.
Shyamala
V
V
Siani
M A
MA
Gallegos
C A
CA
Feucht
P H
PH
Abbott
J
J
Lapointe
G R
GR
Moghadam
M
M
Khoja
H
H
Zakel
J
J
Tekamp-Olson
P
P
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D054360:Chemokine CXCL1; D018925:Chemokines / Q000737:chemistry; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000737:chemistry; D002634:Chemotaxis, Leukocyte; D017931:DNA Primers / Q000737:chemistry; D006133:Growth Substances / Q000737:chemistry; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018123:Receptors, Interleukin / Q000737:chemistry*; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D015703:Antigens, CD; C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D018925:Chemokines; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D017931:DNA Primers; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins