Mutagenesis Studies of Interleukin-8

Williams, Glyn; Borkakoti, Neera; Bottomley, Gillian A.; Cowan, I. C.; Fallowfield, Amanda G.; Jones, Philip S.; Kirtland, Stephen J.; Price, G J; Price, Lauri

doi:10.1074/jbc.271.16.9579

Cited by 69 publications

(44 citation statements)

References 37 publications

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“…Chemokines are divided into four families based on the pattern of cysteine residues (C, CC, CXC, and CX 3 C), and they exert their effects by binding and signaling through seven transmembrane G protein-coupled receptors (GPCRs) on the surface of leukocytes. Although the molecular details remain unclear, mutagenesis and NMR studies have revealed some of the structural requirements for receptor binding and activation (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Collectively, these studies support a hypothesis whereby chemokines activate their receptor through a putative two-step mechanism.…”

Section: T He Directed Migration Of Leukocytes Into Areas Of Inflammasupporting

confidence: 61%

“…Arginine at position 24 is also highly conserved among MCPs and resides within a 3 10 helix present in every known CCchemokine structure (6). Mutation of R24 to alanine (R24A) produced the second largest reduction (17-fold) in the interaction with VV-35kDa ( Table 1, Fig.…”

Section: Vv-35kda Contacts Many Of the Same Residues Of Mcp-1 That Con-mentioning

confidence: 99%

“…2). We also tested a mutant containing a proline substitution for isoleucine at position 5 (I5P), which would be expected to disrupt the putative 3 10 helix observed in the crystal structure of MCP-1 (49), but found no change in binding affinity. A double mutation of V9 and T10 to alanine (V9A͞T10A) also had no effect on binding to VV-35kDa (Table 1), although mutation of T10 to Glu (T10E) produced a 6-fold reduction in affinity, suggesting that the residue at position 10 to may be positioned close to a complementary region on VV-35kDa that is negatively charged.…”

Section: N-terminal Residues Of Mcp-1 Are Not Critical For Binding Tomentioning

confidence: 99%

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Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor

Seet

Singh

Paavola

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Poxviruses express a family of secreted proteins that bind with high affinity to chemokines and antagonize the interaction with their cognate G protein-coupled receptors (GPCRs). These viral inhibitors are novel in structure and, unlike cellular chemokine receptors, are able to specifically interact with most, if not all, CC-chemokines. We therefore sought to define the structural features of CC-chemokines that facilitate this broad-spectrum interaction. Here, we identify the residues present on human monocyte chemoattractant protein-1 (MCP-1) that are required for highaffinity interaction with the vaccinia virus 35-kDa CC-chemokine binding protein (VV-35kDa). Not only do these residues correspond to those required for interaction with the cognate receptor CCR2b but they are also conserved among many CC-chemokines. Thus, the results provide a structural basis for the ability of VV-35kDa to promiscuously recognize CC-chemokines and block binding to their receptors.CCR2b ͉ chemokine binding protein ͉ monocyte chemoattractant protein-1 ͉ mutagenesis ͉ surface plasmon resonance

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Section: T He Directed Migration Of Leukocytes Into Areas Of Inflammasupporting

confidence: 61%

Section: Vv-35kda Contacts Many Of the Same Residues Of Mcp-1 That Con-mentioning

confidence: 99%

Section: N-terminal Residues Of Mcp-1 Are Not Critical For Binding Tomentioning

confidence: 99%

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Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor

Seet

Singh

Paavola

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Studies on the IL-8 structure-function relationship suggested that multiple domains of IL-8 are involved in binding and signaling (12)(13)(14). It has been shown that the interaction domains between the inflammatory protein C5a and its receptor consist of two distinct subsites and the occupation of only one of the subsites is required for receptor activation (35).…”

Section: Discussionmentioning

confidence: 99%

“…Results obtained from mutagenesis and synthetic analogs of IL-8 demonstrated that residues Glu 4-Leu 5-Arg 6 of IL-8 were necessary, but not sufficient, for high affinity binding and activities (9 -12). Recent studies identified a second region, comprising residues that cluster near a surface-accessible hydrophobic pocket, that is important for IL-8 function (13,14). This region contains a second site for IL-8 receptor recognition that, in combination with the Glu 4-Leu 5-Arg 6 region, can modulate receptor binding affinity and specificity.…”

mentioning

confidence: 99%

Discrete Steps in Binding and Signaling of Interleukin-8 with Its Receptor

Ruffing

Shi

et al. 1996

Journal of Biological Chemistry

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The mechanisms by which chemokines bind and signal through their receptors are complex and poorly understood. In the present study, we sought to dissect these processes and to map important functional domains of the two CXC chemokine (interleukin-8) receptors, CXCR1 (formally IL-8RA) and CXCR2 (formally IL-8RB), using blocking monoclonal antibodies (mAbs) to the receptors and a series of chimeras between CXCR1 and CXCR2. A panel of specific mAbs against CXCR1 or CXCR2, generated by immunizing mice with transfectants expressing either receptor, were shown to effectively block IL-8-and/or growth-related oncogene ␣ (GRO␣) -mediated ligand binding, chemotaxis, elastase release, and VCAM-1 binding in CXCR1 and CXCR2 transfectants and/or human neutrophils. Of particular interest was an anti-CXCR1 mAb, 7D9, that inhibited chemotaxis, elastase release, and VCAM-1 binding but had no detectable effects on ligand binding. The epitopes of these blocking mAbs were mapped by using a series of CXCR1/2 chimera transfectants and synthetic peptides. Most of the anti-CXCR1 antibodies, except 7D9, mapped to the amino acid sequence WDFDDL (CXCR1 residues 10 -15), and all the anti-CXCR2 antibodies mapped to the amino acid sequence FEDFW (CXCR2 residues 6 -10). The epitope of mAb 7D9 mainly involved a region within the first 45 residues of CXCR1, and it appeared to be conformation-sensitive. These results support a model in which the binding and signaling of IL-8 with its receptor occur in at least two discrete steps involving distinct domains of the receptor. This model is consistent with the notion that discrete conformational changes of the receptor secondary to ligand binding are required to trigger various biological responses. Moreover, the ligand binding and chemotaxis properties of each CXCR1/2 chimeric receptor to IL-8 and GRO␣ were determined. It was found that each is distinct in its ability to confer ligand binding and chemotactic response to IL-8 and GRO␣, and two conclusions could be made. 1) The N-terminal segment of CXCR1 is a dominant determinant of receptor subtype selectivity, consistent with previous studies using rabbit/human CXCR1/2 chimeras; and 2) the specificity determinant for GRO␣ binding in CXCR2 involves sequences in the N terminus, distal to the first 15 residues, as well as other parts of the receptor.

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Structural change and receptor binding in a chemokine mutant with a rearranged disulfide: X-ray structure of e38C/C50A IL-8 at 2 Å resolution

Lowman

Chee

Artis³

1997

Proteins

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The characteristic CXC chemokine disulfide core of interleukin-8 (IL-8) has been rearranged in a variant replacing the 9-50 disulfide with a 9-38 disulfide. The new variant has been characterized by its binding affinity to IL-8 receptors A and B and the erythrocyte receptor DARC. This variant binds the three receptors with affinities between 500- and 2,500-fold lower than wild-type IL-8. Binding affinity results are also reported for the variant with alanine substituted for both cysteines 9 and 50. The Glu38-->Cys/Cys50-->Ala IL-8 crystallizes in space group P2(1)2(1)2(1) with cell parameters a = 46.4, b = 49.2, and c = 69.5 A, and has been refined to an R-value of 19.4% for data from 10 to 2 A resolution. Analysis of the structure confirms the new disulfide arrangement and suggests that changes at Ile10 may be the principal cause of the lowered affinities.

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Mutagenesis Studies of Interleukin-8

Cited by 69 publications

References 37 publications

Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor

Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor

Discrete Steps in Binding and Signaling of Interleukin-8 with Its Receptor

Structural change and receptor binding in a chemokine mutant with a rearranged disulfide: X-ray structure of e38C/C50A IL-8 at 2 Å resolution

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