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NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding - PubMed

  • ️Sat Jan 01 2000

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

J R Huth et al. Proc Natl Acad Sci U S A. 2000.

Abstract

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal alpha-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal alpha-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

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Figures

Figure 1
Figure 1

(A) Region of the 15N-HSQC spectrum showing an overlay of chemical shifts of 0.2 mM CD11a I domain in the presence (blue) and absence (red) of 0.04 mM ICAM-1 domains 1 and 2. (B) Region of the 13C-constant time HSQC spectrum showing an overlay of chemical shifts of 0.2 mM CD11a I domain in the presence (blue) and absence (red) of 0.1 mM ICAM-1 domains 1 and 2.

Figure 2
Figure 2

Ribbon and surface representations of the x-ray crystal structure of the CD11a I domain (4) showing residues affected by the addition of soluble ICAM-1 domains 1 and 2. Colors indicate the extent of line broadening: red, large decrease in peak height; blue, medium or no decrease in peak height; gray, unassigned in the complex due to spectral overlap. The magnesium ion of the MIDAS is shown in yellow. For the ribbon diagrams, the color coding of the ribbon indicates changes in peak intensity of amide proton signals, and colors of side chains indicate changes in peak intensity of methyl proton signals. For the surface representations, red indicates a large decrease in peak height of 15N-amide or 13C-methyl signals. (A and B) View of α-helix 7 and the opposing β-sheet. (C and D) Opposite face of the I domain showing α-helix 4.

Figure 3
Figure 3

Expression of LFA-1 mutants on COS cells and representative data for the ICAM-1 adhesion assay. Expression of the wild type and a subset of mutants that do not respond to mAb 240Q induction was determined by flow cytofluorometry. Data from a representative experiment are shown and were similar for all mutants and across all experiments. (A) Transfection efficacy, expressed as the percentage of cells staining positive for CD11a (TS1/22) and CD18 (TS1/18 and 240Q). (B) The relative expression levels of different mutants expressed as median MFI of CD11a and CD18 mAb. (C) Adhesion assay of COS cells expressing wild-type LFA-1 or LFA-1 with a D137A mutation in presence of Control mAb (1B7), Blocking mAb (TS1/22), or Activating mAb 240Q. The adhesion assay is representative of greater than seven independent experiments. Mean absorbency values and standard deviations from triplicate wells are shown.

Figure 4
Figure 4

Average adhesion of COS cells expressing LFA-1 I domain mutants to ICAM-1. Filled bars indicate the percent of adhesion that occurs in the presence of ICAM-1. Open bars indicate the percentage of adhesion that occurs in the presence of ICAM-1 and the mAb 240Q. Data presented represent the mean values and standard error of at least three independent experiments. Data were first normalized to BSA and expressed relative to binding of wild-type LFA-1 under the same stimulation condition and within the same assay to compare data from multiple experiments (see Materials and Methods). (A) Alanine mutations that cause no significant change in adhesion both in the presence or absence of mAb 240Q. (B) Mutations that increased adhesion, but whose mAb 240Q response is equivalent to the wild-type response. (C) Mutations that decrease adhesion but whose adhesion is restored to wild-type levels (50–100% of wild type) by mAb 240Q. (D) Mutations that decrease adhesion relative to the wild type but whose effects cannot be reversed by mAb 240Q.

Figure 5
Figure 5

Location of I domain substitution mutations in the IDAS. The color coding indicates the affect on ICAM-1 binding: blue, wild-type levels of adhesion with or without mAb 240Q induction; green, greater than wild-type levels of adhesion without mAb 240Q and wild-type levels with the antibody; yellow, decreased adhesion without mAb 240Q and wild-type adhesion with the antibody; red, decreased adhesion with and without mAb 240Q.

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