Exchanging interleukin-8 and melanoma growth-stimulating activity receptor binding specificities.
Interleukin-8 (IL-8), a CXC chemokine, is known to bring about chemotaxis and activation of neutrophils through high affinity binding to at least two distinct receptors, receptor-A and receptor-B. The IL-8 homolog melanoma growth stimulating activity (MGSA) is also active toward neutrophils. In contrast to IL-8, MGSA binds receptor-B with high affinity and binds receptor-A with approximately 400-fold lower affinity. Using the structure of IL-8 (Clore et al.(1990) Biochemistry, 29, 1689-1696; Baldwin et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 502-506) and the NMR-determined structure of MGSA (Fairbrother et al. (1994) J. Mol. Biol. 242, 252-270), we designed variants of both IL-8 and MGSA to investigate the basis of specificity for binding of these chemokines to the IL-8 receptors. The most outstanding structural difference between IL-8 and MGSA lies in the loop preceding the first beta-strand. When the corresponding (shorter) loop from MGSA was swapped into IL-8, both receptor-A and receptor-B binding affinities were significantly (>300-fold) reduced. However, with additional mutations that affect packing interactions, an IL-8 variant specific for receptor-B binding was produced. Conversely, when the same loop from IL-8 was swapped into MGSA, receptor-B binding was maintained with only a approximately 30-fold reduction in receptor-A affinity. Again, mutations affecting packing of the loop yielded a MGSA variant with high affinity for both receptors, like IL-8. Finally, we show, through point mutations in a monomeric IL-8 framework, that individual side chain substitutions can affect receptor specificity.
Lowman
H B
HB
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Slagle
P H
PH
DeForge
L E
LE
Wirth
C M
CM
Gillece-Castro
B L
BL
Bourell
J H
JH
Fairbrother
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D054360:Chemokine CXCL1; D018925:Chemokines / Q000378:metabolism; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D003198:Computer Simulation; D004926:Escherichia coli; D006133:Growth Substances / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011817:Rabbits; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012984:Software; D013379:Substrate Specificity; D014162:Transfection
D015703:Antigens, CD; C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D018925:Chemokines; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D018121:Receptors, Cytokine; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011994:Recombinant Proteins; C078862:melanoma growth stimulating activity receptor