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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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Effects of mutations involving F302, F275,  and T209 on iC3b, fibrinogen,  and NIF binding to recombinant membrane-bound CR3.  (A) Histograms (mean ±  SEM, n = 3) showing the  normal cell surface expression of the mutant CR3 on  transfected COS cells, as  checked by anti-CD11b mAb  903 and anti-CD18 mAb TS1/ 18. (B) Histograms (mean ±  SEM, n = 4) showing the  binding of mutant CR3 expressed on COS cells to iC3b and NIF. (C) Histograms (mean ± SEM, n = 3) showing the binding of WT or  F302W CR3-transfected CHO cells to iC3b and fibrinogen (Fg). The surface expression levels of both WT and F302W receptors were  ∼70% (data not shown).
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Figure 4: Effects of mutations involving F302, F275, and T209 on iC3b, fibrinogen, and NIF binding to recombinant membrane-bound CR3. (A) Histograms (mean ± SEM, n = 3) showing the normal cell surface expression of the mutant CR3 on transfected COS cells, as checked by anti-CD11b mAb 903 and anti-CD18 mAb TS1/ 18. (B) Histograms (mean ± SEM, n = 4) showing the binding of mutant CR3 expressed on COS cells to iC3b and NIF. (C) Histograms (mean ± SEM, n = 3) showing the binding of WT or F302W CR3-transfected CHO cells to iC3b and fibrinogen (Fg). The surface expression levels of both WT and F302W receptors were ∼70% (data not shown).

Mentions: A second structural difference between the open and closed r11bA conformations is a major increase in the solvent accessibility of the conserved F302 and F275 in the open conformation (relative total side chain accessibility values increase from 6.4 to 81.5% for F302 and from 0.3 and 26.4% for F275). Therefore, we assessed the effects of introducing bulky or charged side chains at these positions on the activation-dependent interaction of CR3 with iC3b. F302 was replaced with arginine, tryptophan, or tyrosine (as a control), and F275 was replaced with arginine. The CR3 mutant F302R was not expressed on the cell surface (data not shown). The remaining mutants were surface expressed in normal quantities relative to the WT receptor (Fig. 4 A). To determine the functional profile of the resulting mutants, the recombinant receptors were probed with the activation-dependent and -independent ligands iC3b and NIF, respectively. Whereas the binding of iC3b to the conserved F302Y receptor was indistinguishable from that of WT, iC3b binding to F302W and F275R was significantly increased (Fig. 4 B). On the other hand, NIF bound equally to F302Y, F302W, and F275R receptors. Similar observations were obtained in stable CHO cell lines expressing comparable amounts of WT or F302W (Fig. 4 C). Increased binding of F302W was not limited to iC3b, but also occurred with fibrinogen, another activation-dependent ligand (Fig. 4 C).


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)

Effects of mutations involving F302, F275,  and T209 on iC3b, fibrinogen,  and NIF binding to recombinant membrane-bound CR3.  (A) Histograms (mean ±  SEM, n = 3) showing the  normal cell surface expression of the mutant CR3 on  transfected COS cells, as  checked by anti-CD11b mAb  903 and anti-CD18 mAb TS1/ 18. (B) Histograms (mean ±  SEM, n = 4) showing the  binding of mutant CR3 expressed on COS cells to iC3b and NIF. (C) Histograms (mean ± SEM, n = 3) showing the binding of WT or  F302W CR3-transfected CHO cells to iC3b and fibrinogen (Fg). The surface expression levels of both WT and F302W receptors were  ∼70% (data not shown).
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getmorefigures.php?uid=PMC2132978&req=5

Figure 4: Effects of mutations involving F302, F275, and T209 on iC3b, fibrinogen, and NIF binding to recombinant membrane-bound CR3. (A) Histograms (mean ± SEM, n = 3) showing the normal cell surface expression of the mutant CR3 on transfected COS cells, as checked by anti-CD11b mAb 903 and anti-CD18 mAb TS1/ 18. (B) Histograms (mean ± SEM, n = 4) showing the binding of mutant CR3 expressed on COS cells to iC3b and NIF. (C) Histograms (mean ± SEM, n = 3) showing the binding of WT or F302W CR3-transfected CHO cells to iC3b and fibrinogen (Fg). The surface expression levels of both WT and F302W receptors were ∼70% (data not shown).
Mentions: A second structural difference between the open and closed r11bA conformations is a major increase in the solvent accessibility of the conserved F302 and F275 in the open conformation (relative total side chain accessibility values increase from 6.4 to 81.5% for F302 and from 0.3 and 26.4% for F275). Therefore, we assessed the effects of introducing bulky or charged side chains at these positions on the activation-dependent interaction of CR3 with iC3b. F302 was replaced with arginine, tryptophan, or tyrosine (as a control), and F275 was replaced with arginine. The CR3 mutant F302R was not expressed on the cell surface (data not shown). The remaining mutants were surface expressed in normal quantities relative to the WT receptor (Fig. 4 A). To determine the functional profile of the resulting mutants, the recombinant receptors were probed with the activation-dependent and -independent ligands iC3b and NIF, respectively. Whereas the binding of iC3b to the conserved F302Y receptor was indistinguishable from that of WT, iC3b binding to F302W and F275R was significantly increased (Fig. 4 B). On the other hand, NIF bound equally to F302Y, F302W, and F275R receptors. Similar observations were obtained in stable CHO cell lines expressing comparable amounts of WT or F302W (Fig. 4 C). Increased binding of F302W was not limited to iC3b, but also occurred with fibrinogen, another activation-dependent ligand (Fig. 4 C).

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