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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 A-domain  mutants on CR3 surface expression and iC3b binding.  (A) Histograms (mean ±  SEM, n = 3) showing the relative binding of anti-CD11b  mAb OKM10 to COS cells  expressing mutant CR3. (B)  Histograms (mean ± SEM,  n = 3) showing the relative  binding of iC3b to COS cells  expressing CR3 mutants. (Inset) Radioautograph of a  Western blot showing comparable amounts of the  CD11b subunit in anti-CD18  immunoprecipitates from  COS cells expressing WT or  mutant CR3. No CD11b was  seen in anti-CD18 immunoprecipitates from COS cells  transfected with CD18  cDNA alone.
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Figure 2: Effects of A-domain mutants on CR3 surface expression and iC3b binding. (A) Histograms (mean ± SEM, n = 3) showing the relative binding of anti-CD11b mAb OKM10 to COS cells expressing mutant CR3. (B) Histograms (mean ± SEM, n = 3) showing the relative binding of iC3b to COS cells expressing CR3 mutants. (Inset) Radioautograph of a Western blot showing comparable amounts of the CD11b subunit in anti-CD18 immunoprecipitates from COS cells expressing WT or mutant CR3. No CD11b was seen in anti-CD18 immunoprecipitates from COS cells transfected with CD18 cDNA alone.

Mentions: We introduced single or double amino acid substitutions in residues scattered in all 11 loops and adjacent helices of the r11bA structure with the exception of the largely hydrophobic βC-α2 loop (H183FT) and the α2-α3 loop (N192PNP) (where a P195A substitution had detrimental effects on metal ion coordination; Michishita et al., 1993). All of the targeted residues had solvent-exposed side chains (relative total side chain accessibility values ranged from ∼25 to 100%), and were replaced either with residues from CD11a (which does not bind to iC3b or NIF), with residues having the opposite charge, or with alanines. The WT and mutant receptors expressed in COS cells were then probed with mAbs or ligands as described (Michishita et al., 1993; Rieu et al., 1996). None of the mutations affected the normal expression of the receptors as judged by binding of the anti-CD11b mAbs OKM10 (Fig. 2 A), 903, and the anti-CD18 mAb TS1/18 (Rieu et al., 1996; and data not shown). Binding of G143M, D149K, E178E/AA, T203Q/KH, R208L, F246K, and E278K/AA receptors to iC3b was either absent or significantly reduced (Fig. 2 B), whereas that of E244K and D273K was significantly increased. Gain-of-function mutations are not unusual in contact regions of protein–protein complexes (Clackson and Wells, 1995). A smaller but still significant increase in binding of K166S/AA was also observed. None of the remaining eight mutations (involving 11 amino acids) affected iC3b binding significantly. The loss of iC3b binding to some mutants was not caused by defective formation of the heterodimer (Fig. 2 B, inset; Rieu et al., 1996) or an inability of the domain to bind metal: Mn54 bound directly to G143M, D149K, E178E/AA, and R208L r11bA mutants, and the metal-dependent interaction of biotinylated NIF with the remaining mutants T203Q/KH, E244K, F246K, D273K, and E278K/AA was normal (Rieu et al., 1996). With the exception of E278K and T203 (PQ and K, respectively, in mouse), the residues involved in iC3b binding were either identical (G143, D149, E179, Q204, R208, E244, and F246) or conserved (E178 and D273) in mouse CR3 (Pytela, 1988). This may explain the ability of mouse and human CR3 to bind to human and mouse iC3b, respectively. The relative affinity of these cross-species interactions has not been determined.


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 A-domain  mutants on CR3 surface expression and iC3b binding.  (A) Histograms (mean ±  SEM, n = 3) showing the relative binding of anti-CD11b  mAb OKM10 to COS cells  expressing mutant CR3. (B)  Histograms (mean ± SEM,  n = 3) showing the relative  binding of iC3b to COS cells  expressing CR3 mutants. (Inset) Radioautograph of a  Western blot showing comparable amounts of the  CD11b subunit in anti-CD18  immunoprecipitates from  COS cells expressing WT or  mutant CR3. No CD11b was  seen in anti-CD18 immunoprecipitates from COS cells  transfected with CD18  cDNA alone.
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Related In: Results  -  Collection

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Figure 2: Effects of A-domain mutants on CR3 surface expression and iC3b binding. (A) Histograms (mean ± SEM, n = 3) showing the relative binding of anti-CD11b mAb OKM10 to COS cells expressing mutant CR3. (B) Histograms (mean ± SEM, n = 3) showing the relative binding of iC3b to COS cells expressing CR3 mutants. (Inset) Radioautograph of a Western blot showing comparable amounts of the CD11b subunit in anti-CD18 immunoprecipitates from COS cells expressing WT or mutant CR3. No CD11b was seen in anti-CD18 immunoprecipitates from COS cells transfected with CD18 cDNA alone.
Mentions: We introduced single or double amino acid substitutions in residues scattered in all 11 loops and adjacent helices of the r11bA structure with the exception of the largely hydrophobic βC-α2 loop (H183FT) and the α2-α3 loop (N192PNP) (where a P195A substitution had detrimental effects on metal ion coordination; Michishita et al., 1993). All of the targeted residues had solvent-exposed side chains (relative total side chain accessibility values ranged from ∼25 to 100%), and were replaced either with residues from CD11a (which does not bind to iC3b or NIF), with residues having the opposite charge, or with alanines. The WT and mutant receptors expressed in COS cells were then probed with mAbs or ligands as described (Michishita et al., 1993; Rieu et al., 1996). None of the mutations affected the normal expression of the receptors as judged by binding of the anti-CD11b mAbs OKM10 (Fig. 2 A), 903, and the anti-CD18 mAb TS1/18 (Rieu et al., 1996; and data not shown). Binding of G143M, D149K, E178E/AA, T203Q/KH, R208L, F246K, and E278K/AA receptors to iC3b was either absent or significantly reduced (Fig. 2 B), whereas that of E244K and D273K was significantly increased. Gain-of-function mutations are not unusual in contact regions of protein–protein complexes (Clackson and Wells, 1995). A smaller but still significant increase in binding of K166S/AA was also observed. None of the remaining eight mutations (involving 11 amino acids) affected iC3b binding significantly. The loss of iC3b binding to some mutants was not caused by defective formation of the heterodimer (Fig. 2 B, inset; Rieu et al., 1996) or an inability of the domain to bind metal: Mn54 bound directly to G143M, D149K, E178E/AA, and R208L r11bA mutants, and the metal-dependent interaction of biotinylated NIF with the remaining mutants T203Q/KH, E244K, F246K, D273K, and E278K/AA was normal (Rieu et al., 1996). With the exception of E278K and T203 (PQ and K, respectively, in mouse), the residues involved in iC3b binding were either identical (G143, D149, E179, Q204, R208, E244, and F246) or conserved (E178 and D273) in mouse CR3 (Pytela, 1988). This may explain the ability of mouse and human CR3 to bind to human and mouse iC3b, respectively. The relative affinity of these cross-species interactions has not been determined.

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