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EM structure of the ectodomain of integrin CD11b/CD18 and localization of its ligand-binding site relative to the plasma membrane.

Adair BD, Xiong JP, Alonso JL, Hyman BT, Arnaout MA - PLoS ONE (2013)

Bottom Line: One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS).We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state.Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, United States of America.

ABSTRACT
One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state. A well defined density for αA was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that αA is restricted to a relatively small range of orientations relative to the Propeller domain. Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin. These studies are the first to define the 3D EM structure of an αA-containing integrin ectodomain and to position the ligand-binding face of αA domain in relation to the plasma membrane, providing new insights into current models of integrin activation.

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Surface shaded density of the final 3D map of the CD11b/CD18 ectodomain.A, B) are two views of the map rotated by 45°. The isosurface has been chosen to enclose 100% of the expected protein volume. C and D) the same views of the map as in A and B, shown as a transparent isosurface, but also displaying the αA-lacking αVβ3 ectodomain (pdb 3IJE) model as a ribbon diagram. A clear density corresponding to the CD11bA domain is seen, fitted here with a ribbon diagram of the crystal structure of the isolated CD11bA (pdb 1jlm), with the MIDAS ion in cyan. The αV subunit and CD11bA are shown in blue and β3-subunit in red. The spherical metal ions in the Propeller and α-genu are in green and that in the βA ADMIDAS is in magenta. E, F) a ribbon diagram of the crystal structure of CD11c/CD18 ectodomain fitted into the CD11b/CD18 EM map (shown as a transparent isosurface). Most of Calf-1/Calf-2 and β TD domains do not fit the 3D map. CD11cA fits better within the map density but extends somewhat outside it (compare CD11cA with that of CD11bA [shown in yellow, oriented as in C, with its MIDAS metal ion in orange]). The three metal ions in the CD11c Propeller are in green, the CD11cA MIDAS ion in cyan, and the βA ADMIDAS ion in magenta. F) a 45° clockwise rotation of the Figure in (E).
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pone-0057951-g002: Surface shaded density of the final 3D map of the CD11b/CD18 ectodomain.A, B) are two views of the map rotated by 45°. The isosurface has been chosen to enclose 100% of the expected protein volume. C and D) the same views of the map as in A and B, shown as a transparent isosurface, but also displaying the αA-lacking αVβ3 ectodomain (pdb 3IJE) model as a ribbon diagram. A clear density corresponding to the CD11bA domain is seen, fitted here with a ribbon diagram of the crystal structure of the isolated CD11bA (pdb 1jlm), with the MIDAS ion in cyan. The αV subunit and CD11bA are shown in blue and β3-subunit in red. The spherical metal ions in the Propeller and α-genu are in green and that in the βA ADMIDAS is in magenta. E, F) a ribbon diagram of the crystal structure of CD11c/CD18 ectodomain fitted into the CD11b/CD18 EM map (shown as a transparent isosurface). Most of Calf-1/Calf-2 and β TD domains do not fit the 3D map. CD11cA fits better within the map density but extends somewhat outside it (compare CD11cA with that of CD11bA [shown in yellow, oriented as in C, with its MIDAS metal ion in orange]). The three metal ions in the CD11c Propeller are in green, the CD11cA MIDAS ion in cyan, and the βA ADMIDAS ion in magenta. F) a 45° clockwise rotation of the Figure in (E).

Mentions: The resulting map from the final round of refinement is shown in Figure 2A–D. The map has been filtered to 26Å, and the isosurface set to enclose 100% of the expected protein volume. Excluding the density attributable to the CD11bA domain, the map resembles the canonical ‘bent’ conformation as seen in the αVβ3 ectodomain structure [25]. The map density does not fit well the X-ray structure for CD11c/CD18 ectodomain (Figure 2E, F): While the head, Thigh and Hybrid domains fit well into the map density, Calf-1/Calf-2 clearly lie outside it (Figure 2E, F). Both ectodomains contain an engineered interchain disulfide bond at the identical position in the membrane-proximal α/β linker region of the ectodomain, so the difference is more likely the result of crystal contacts among the four molecules in the unit cell of CD11c/CD18 ectodomain distorting these domains from their position in solution. The αA domain from the CD11c structure fits better within the map density but extends somewhat outside it (Figure 2E, F), requiring a small 5Å translation from its position in the CD11c X-ray structure [17].


EM structure of the ectodomain of integrin CD11b/CD18 and localization of its ligand-binding site relative to the plasma membrane.

Adair BD, Xiong JP, Alonso JL, Hyman BT, Arnaout MA - PLoS ONE (2013)

Surface shaded density of the final 3D map of the CD11b/CD18 ectodomain.A, B) are two views of the map rotated by 45°. The isosurface has been chosen to enclose 100% of the expected protein volume. C and D) the same views of the map as in A and B, shown as a transparent isosurface, but also displaying the αA-lacking αVβ3 ectodomain (pdb 3IJE) model as a ribbon diagram. A clear density corresponding to the CD11bA domain is seen, fitted here with a ribbon diagram of the crystal structure of the isolated CD11bA (pdb 1jlm), with the MIDAS ion in cyan. The αV subunit and CD11bA are shown in blue and β3-subunit in red. The spherical metal ions in the Propeller and α-genu are in green and that in the βA ADMIDAS is in magenta. E, F) a ribbon diagram of the crystal structure of CD11c/CD18 ectodomain fitted into the CD11b/CD18 EM map (shown as a transparent isosurface). Most of Calf-1/Calf-2 and β TD domains do not fit the 3D map. CD11cA fits better within the map density but extends somewhat outside it (compare CD11cA with that of CD11bA [shown in yellow, oriented as in C, with its MIDAS metal ion in orange]). The three metal ions in the CD11c Propeller are in green, the CD11cA MIDAS ion in cyan, and the βA ADMIDAS ion in magenta. F) a 45° clockwise rotation of the Figure in (E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585415&req=5

pone-0057951-g002: Surface shaded density of the final 3D map of the CD11b/CD18 ectodomain.A, B) are two views of the map rotated by 45°. The isosurface has been chosen to enclose 100% of the expected protein volume. C and D) the same views of the map as in A and B, shown as a transparent isosurface, but also displaying the αA-lacking αVβ3 ectodomain (pdb 3IJE) model as a ribbon diagram. A clear density corresponding to the CD11bA domain is seen, fitted here with a ribbon diagram of the crystal structure of the isolated CD11bA (pdb 1jlm), with the MIDAS ion in cyan. The αV subunit and CD11bA are shown in blue and β3-subunit in red. The spherical metal ions in the Propeller and α-genu are in green and that in the βA ADMIDAS is in magenta. E, F) a ribbon diagram of the crystal structure of CD11c/CD18 ectodomain fitted into the CD11b/CD18 EM map (shown as a transparent isosurface). Most of Calf-1/Calf-2 and β TD domains do not fit the 3D map. CD11cA fits better within the map density but extends somewhat outside it (compare CD11cA with that of CD11bA [shown in yellow, oriented as in C, with its MIDAS metal ion in orange]). The three metal ions in the CD11c Propeller are in green, the CD11cA MIDAS ion in cyan, and the βA ADMIDAS ion in magenta. F) a 45° clockwise rotation of the Figure in (E).
Mentions: The resulting map from the final round of refinement is shown in Figure 2A–D. The map has been filtered to 26Å, and the isosurface set to enclose 100% of the expected protein volume. Excluding the density attributable to the CD11bA domain, the map resembles the canonical ‘bent’ conformation as seen in the αVβ3 ectodomain structure [25]. The map density does not fit well the X-ray structure for CD11c/CD18 ectodomain (Figure 2E, F): While the head, Thigh and Hybrid domains fit well into the map density, Calf-1/Calf-2 clearly lie outside it (Figure 2E, F). Both ectodomains contain an engineered interchain disulfide bond at the identical position in the membrane-proximal α/β linker region of the ectodomain, so the difference is more likely the result of crystal contacts among the four molecules in the unit cell of CD11c/CD18 ectodomain distorting these domains from their position in solution. The αA domain from the CD11c structure fits better within the map density but extends somewhat outside it (Figure 2E, F), requiring a small 5Å translation from its position in the CD11c X-ray structure [17].

Bottom Line: One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS).We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state.Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, United States of America.

ABSTRACT
One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state. A well defined density for αA was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that αA is restricted to a relatively small range of orientations relative to the Propeller domain. Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin. These studies are the first to define the 3D EM structure of an αA-containing integrin ectodomain and to position the ligand-binding face of αA domain in relation to the plasma membrane, providing new insights into current models of integrin activation.

Show MeSH
Related in: MedlinePlus