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Two functional states of the CD11b A-domain: correlations with key features of two Mn2+-complexed crystal structures.

Li R, Rieu P, Griffith DL, Scott D, Arnaout MA - J. Cell Biol. (1998)

Bottom Line: Approximately 10% of wild-type CD11b A-domain is present in an "active" state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3).In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively.The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures.

View Article: PubMed Central - PubMed

Affiliation: Leukocyte Biology and Inflammation Program, Renal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.

ABSTRACT
In the presence of bound Mn2+, the three- dimensional structure of the ligand-binding A-domain from the integrin CR3 (CD11b/CD18) is shown to exist in the "open" conformation previously described only for a crystalline Mg2+ complex. The open conformation is distinguished from the "closed" form by the solvent exposure of F302, a direct T209-Mn2+ bond, and the presence of a glutamate side chain in the MIDAS site. Approximately 10% of wild-type CD11b A-domain is present in an "active" state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3). In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively. The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures. We suggest that stabilization of the open structure is independent of the nature of the metal ligand and that the open conformation may represent the physiologically active form.

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Interaction of mAb 7E3 with WT or F302W-transfected CHO cells. Expression of the activation-specific 7E3 epitope on WT, F302W, or Neo-transfected CHO cells was examined by flow cytometry. mAb 44, which recognizes the whole  population of CR3, was used as a reference. Mouse IgG1 (dotted  lines) was used to determine background fluorescence. The fraction of CR3 recognized by 7E3 is 5.95 ± 1.16% for WT and 14.9 ±  3.47% for F302W (n = 6).
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Figure 5: Interaction of mAb 7E3 with WT or F302W-transfected CHO cells. Expression of the activation-specific 7E3 epitope on WT, F302W, or Neo-transfected CHO cells was examined by flow cytometry. mAb 44, which recognizes the whole population of CR3, was used as a reference. Mouse IgG1 (dotted lines) was used to determine background fluorescence. The fraction of CR3 recognized by 7E3 is 5.95 ± 1.16% for WT and 14.9 ± 3.47% for F302W (n = 6).

Mentions: The 11bA recognizing mAb 7E3 (Zhou et al., 1994) is a ligand-mimetic antibody whose binding to the holoreceptor in leukocytes or in cells transfected with recombinant CR3 is stimulated by the same agonists that trigger physiologic ligand binding (Altieri and Edgington, 1988; Altieri, 1991; Simon et al., 1997). 7E3 is thus very useful in detecting high affinity CR3 in intact cells. The estimate of the later species is ∼10% when 7E3 or a second ligand mimetic mAb CBRM1/5 (Diamond and Springer, 1993; Simon et al., 1997) is used. To assess the impact of the F302W modification on affinity modulation in CR3, we compared the binding of 7E3 to CHO cells expressing WT or F302W CR3. In six independent experiments (one of which is shown in Fig. 5), we found that the fraction of CR3 recognized by 7E3 was 5.95 ± 1.6% of total CR3 for WT and 14.9 ± 3.47% for F302W. Thus the F302W substitution increases the fraction of high affinity CR3 in whole cells. Increased binding of iC3b and 7E3 to F302W r11bA was also observed (Fig. 6), whereas binding of NIF was unchanged. Taken together, the above findings indicate that a conformational change intrinsic to the A-domain enhances the binding of the holoreceptor as well as the isolated A-domain to activation-dependent ligands. Since 7E3 is a marker of the high affinity state, the observed increase in receptor binding appears to largely result from a change in receptor affinity.


Two functional states of the CD11b A-domain: correlations with key features of two Mn2+-complexed crystal structures.

Li R, Rieu P, Griffith DL, Scott D, Arnaout MA - J. Cell Biol. (1998)

Interaction of mAb 7E3 with WT or F302W-transfected CHO cells. Expression of the activation-specific 7E3 epitope on WT, F302W, or Neo-transfected CHO cells was examined by flow cytometry. mAb 44, which recognizes the whole  population of CR3, was used as a reference. Mouse IgG1 (dotted  lines) was used to determine background fluorescence. The fraction of CR3 recognized by 7E3 is 5.95 ± 1.16% for WT and 14.9 ±  3.47% for F302W (n = 6).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132978&req=5

Figure 5: Interaction of mAb 7E3 with WT or F302W-transfected CHO cells. Expression of the activation-specific 7E3 epitope on WT, F302W, or Neo-transfected CHO cells was examined by flow cytometry. mAb 44, which recognizes the whole population of CR3, was used as a reference. Mouse IgG1 (dotted lines) was used to determine background fluorescence. The fraction of CR3 recognized by 7E3 is 5.95 ± 1.16% for WT and 14.9 ± 3.47% for F302W (n = 6).
Mentions: The 11bA recognizing mAb 7E3 (Zhou et al., 1994) is a ligand-mimetic antibody whose binding to the holoreceptor in leukocytes or in cells transfected with recombinant CR3 is stimulated by the same agonists that trigger physiologic ligand binding (Altieri and Edgington, 1988; Altieri, 1991; Simon et al., 1997). 7E3 is thus very useful in detecting high affinity CR3 in intact cells. The estimate of the later species is ∼10% when 7E3 or a second ligand mimetic mAb CBRM1/5 (Diamond and Springer, 1993; Simon et al., 1997) is used. To assess the impact of the F302W modification on affinity modulation in CR3, we compared the binding of 7E3 to CHO cells expressing WT or F302W CR3. In six independent experiments (one of which is shown in Fig. 5), we found that the fraction of CR3 recognized by 7E3 was 5.95 ± 1.6% of total CR3 for WT and 14.9 ± 3.47% for F302W. Thus the F302W substitution increases the fraction of high affinity CR3 in whole cells. Increased binding of iC3b and 7E3 to F302W r11bA was also observed (Fig. 6), whereas binding of NIF was unchanged. Taken together, the above findings indicate that a conformational change intrinsic to the A-domain enhances the binding of the holoreceptor as well as the isolated A-domain to activation-dependent ligands. Since 7E3 is a marker of the high affinity state, the observed increase in receptor binding appears to largely result from a change in receptor affinity.

Bottom Line: Approximately 10% of wild-type CD11b A-domain is present in an "active" state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3).In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively.The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures.

View Article: PubMed Central - PubMed

Affiliation: Leukocyte Biology and Inflammation Program, Renal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.

ABSTRACT
In the presence of bound Mn2+, the three- dimensional structure of the ligand-binding A-domain from the integrin CR3 (CD11b/CD18) is shown to exist in the "open" conformation previously described only for a crystalline Mg2+ complex. The open conformation is distinguished from the "closed" form by the solvent exposure of F302, a direct T209-Mn2+ bond, and the presence of a glutamate side chain in the MIDAS site. Approximately 10% of wild-type CD11b A-domain is present in an "active" state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3). In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively. The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures. We suggest that stabilization of the open structure is independent of the nature of the metal ligand and that the open conformation may represent the physiologically active form.

Show MeSH
Related in: MedlinePlus