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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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Equilibrium analysis of the interaction of WT or F302W A-domains with immobilized iC3b. Concentrations ranging from 0.5  to15 μM for WT (A) and 0.5 to 12 μM for F302W (B) were injected at a flow rate of 5 μl/min for 6 min to reach equilibrium. C (for WT)  and D (for F302W) show the respective saturable binding curves and Scatchard plots (insets).
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Figure 8: Equilibrium analysis of the interaction of WT or F302W A-domains with immobilized iC3b. Concentrations ranging from 0.5 to15 μM for WT (A) and 0.5 to 12 μM for F302W (B) were injected at a flow rate of 5 μl/min for 6 min to reach equilibrium. C (for WT) and D (for F302W) show the respective saturable binding curves and Scatchard plots (insets).

Mentions: A simple model that explains the above functional and structural studies is that the isolated r11bA exists in two functional states in solution, one active (defined by its ability to bind to activation-dependent ligands) and the other inactive (although still able to bind certain antagonists such as NIF). Activating mutations (such as F302W) or inactivating mutations (such as T209A) change the relative abundance of these two states. To test this model, we used surface plasmon resonance (Malmqvist, 1993) and examined the binding of WT and F302W r11bA to iC3b and mAb 904, an activation-independent and metal-independent ligand. When 1 μM of WT and F302W was injected onto the BIAcore™ sensor chip coupled to excess iC3b, an approximately twofold increase in binding of F302W was observed compared with WT (Fig. 7 A), in agreement with the other binding data presented in Figs. 4–6. When the same amounts of WT and F302W were injected onto the mAb 904–coupled chip surface, no difference in the binding level was found (Fig. 7 B). This indicates that equivalent amounts of WT and F302W r11bA were available for binding. Injection of increasing concentrations of the WT r11bA ranging from 0.5 to 15 μM at a flow rate of 5 μl/min gave a saturable binding curve (Fig. 8, A and C). Scatchard plot of the binding data was linear, with a dissociation constant Kd of 3.8 μM (Fig. 8 C). A similar analysis using F302W revealed an almost identical affinity (Fig. 8, B and D). The observed ligand-binding affinity was significantly lower than that reported for binding of purified CR3 to ligands (Kd ∼12.5–200 nM) (Berman et al., 1993; Cai and Wright, 1995). One interpretation for this difference is that the A-domain preparation contains only a subpopulation of active species. Biosensor technology can be used to measure the active analyte in a protein preparation under conditions where binding to ligand (present in excess) is only limited by the diffusion of the analyte to the surface-bound ligand. Under these conditions, initial binding rates are proportional to the active analyte concentration and independent of the analyte-ligand affinity. This can be validated experimentally by demonstrating that the initial binding rate is independent of ligand density (Karlsson et al., 1993). We used mAb 904 in parallel in order to estimate total binding. By decreasing the flow rate and increasing the ligand density, the binding rate of WT and F302W r11bA to chips coated with iC3b or mAb 904 can be made independent of the respective ligand density (Table III) and therefore a function of the concentration of the binding active species. In WT, this active species represented ∼11 ± 1.7% of the total r11bA (Table IV). In parallel experiments, we showed that the proportion of the active species in F302W increased by ∼2.5-fold (25 ± 1.1%). The fact that a major portion of F302W remains in the inactive state probably reflects other structural considerations that allow motion of the α7 helix to that of the closed form, despite a partial burial of the side chain at position 302. The recent structure of the CD49b A-domain shows that this does in fact occur: the orientation of the α7 helix is very similar to that in the closed form of r11bA, despite only a partial burial of the side chain of E318 (equivalent to F302) at the top of the α7 helix (Emsley et al., 1997). Since, the total amount of r11bA was used in calculating the binding affinities shown in Fig. 8, the affinity of the active species should be ∼10-fold higher, approaching that calculated for purified CR3 and of the active cell-bound form of CD11a/CD18 (Lollo et al., 1993).


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)

Equilibrium analysis of the interaction of WT or F302W A-domains with immobilized iC3b. Concentrations ranging from 0.5  to15 μM for WT (A) and 0.5 to 12 μM for F302W (B) were injected at a flow rate of 5 μl/min for 6 min to reach equilibrium. C (for WT)  and D (for F302W) show the respective saturable binding curves and Scatchard plots (insets).
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Related In: Results  -  Collection

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Figure 8: Equilibrium analysis of the interaction of WT or F302W A-domains with immobilized iC3b. Concentrations ranging from 0.5 to15 μM for WT (A) and 0.5 to 12 μM for F302W (B) were injected at a flow rate of 5 μl/min for 6 min to reach equilibrium. C (for WT) and D (for F302W) show the respective saturable binding curves and Scatchard plots (insets).
Mentions: A simple model that explains the above functional and structural studies is that the isolated r11bA exists in two functional states in solution, one active (defined by its ability to bind to activation-dependent ligands) and the other inactive (although still able to bind certain antagonists such as NIF). Activating mutations (such as F302W) or inactivating mutations (such as T209A) change the relative abundance of these two states. To test this model, we used surface plasmon resonance (Malmqvist, 1993) and examined the binding of WT and F302W r11bA to iC3b and mAb 904, an activation-independent and metal-independent ligand. When 1 μM of WT and F302W was injected onto the BIAcore™ sensor chip coupled to excess iC3b, an approximately twofold increase in binding of F302W was observed compared with WT (Fig. 7 A), in agreement with the other binding data presented in Figs. 4–6. When the same amounts of WT and F302W were injected onto the mAb 904–coupled chip surface, no difference in the binding level was found (Fig. 7 B). This indicates that equivalent amounts of WT and F302W r11bA were available for binding. Injection of increasing concentrations of the WT r11bA ranging from 0.5 to 15 μM at a flow rate of 5 μl/min gave a saturable binding curve (Fig. 8, A and C). Scatchard plot of the binding data was linear, with a dissociation constant Kd of 3.8 μM (Fig. 8 C). A similar analysis using F302W revealed an almost identical affinity (Fig. 8, B and D). The observed ligand-binding affinity was significantly lower than that reported for binding of purified CR3 to ligands (Kd ∼12.5–200 nM) (Berman et al., 1993; Cai and Wright, 1995). One interpretation for this difference is that the A-domain preparation contains only a subpopulation of active species. Biosensor technology can be used to measure the active analyte in a protein preparation under conditions where binding to ligand (present in excess) is only limited by the diffusion of the analyte to the surface-bound ligand. Under these conditions, initial binding rates are proportional to the active analyte concentration and independent of the analyte-ligand affinity. This can be validated experimentally by demonstrating that the initial binding rate is independent of ligand density (Karlsson et al., 1993). We used mAb 904 in parallel in order to estimate total binding. By decreasing the flow rate and increasing the ligand density, the binding rate of WT and F302W r11bA to chips coated with iC3b or mAb 904 can be made independent of the respective ligand density (Table III) and therefore a function of the concentration of the binding active species. In WT, this active species represented ∼11 ± 1.7% of the total r11bA (Table IV). In parallel experiments, we showed that the proportion of the active species in F302W increased by ∼2.5-fold (25 ± 1.1%). The fact that a major portion of F302W remains in the inactive state probably reflects other structural considerations that allow motion of the α7 helix to that of the closed form, despite a partial burial of the side chain at position 302. The recent structure of the CD49b A-domain shows that this does in fact occur: the orientation of the α7 helix is very similar to that in the closed form of r11bA, despite only a partial burial of the side chain of E318 (equivalent to F302) at the top of the α7 helix (Emsley et al., 1997). Since, the total amount of r11bA was used in calculating the binding affinities shown in Fig. 8, the affinity of the active species should be ∼10-fold higher, approaching that calculated for purified CR3 and of the active cell-bound form of CD11a/CD18 (Lollo et al., 1993).

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