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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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EM analysis of the CD11b/CD18-Fab107 complex and orientation of the CD11bA MIDAS face.(A) Reference-free alignment and averaging of CD11b/CD18-Fab107 particles. The top row shows a k-means classification of 10,661 raw particles classified into 10 groups and averaged. The number of particles for each class is indicated at the top of each average. Several of the groups are similar, suggesting that there are fewer than 10 unique preferred orientations for the particles on the grid. The bottom row shows the model projection that best fits each of the particles shown in the top row. Projections in the bottom row were generated from the model shown in (B) which was generated by sequentially fitting a projection for the integrin followed by the projection for the CD11bA/Fab107 complex. For comparison, the model generated by fitting the CD11c/CD18 ectodomain X-ray structure is shown in (C). In this model, the CD11bA domain has the same relative orientation to the rest of the integrin as found in the X-ray structure. (D) Shows a direct comparison of the two models for the average, which most easily allows identification of individual integrin domains within the projection. Displayed alongside the average (center image) is the projection from the model in (B) which best fits the average (left image), compared to the projection from the model in (C) (right image). E, ribbon diagram of the Propeller/αA of CD11c/CD18 ectodomain crystal structure oriented as in Figure 2E, showing crystal contacts from two symmetry-related molecules (Calf-2 in gray and Thigh in magenta) that rotate MIDAS face of CD11cA domain by ∼35° (red arrows) relative to its position in the unrestricted EM model (colored as in Figure 2E). F, same Figure in (E) rotated clockwise by 135°.
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pone-0057951-g003: EM analysis of the CD11b/CD18-Fab107 complex and orientation of the CD11bA MIDAS face.(A) Reference-free alignment and averaging of CD11b/CD18-Fab107 particles. The top row shows a k-means classification of 10,661 raw particles classified into 10 groups and averaged. The number of particles for each class is indicated at the top of each average. Several of the groups are similar, suggesting that there are fewer than 10 unique preferred orientations for the particles on the grid. The bottom row shows the model projection that best fits each of the particles shown in the top row. Projections in the bottom row were generated from the model shown in (B) which was generated by sequentially fitting a projection for the integrin followed by the projection for the CD11bA/Fab107 complex. For comparison, the model generated by fitting the CD11c/CD18 ectodomain X-ray structure is shown in (C). In this model, the CD11bA domain has the same relative orientation to the rest of the integrin as found in the X-ray structure. (D) Shows a direct comparison of the two models for the average, which most easily allows identification of individual integrin domains within the projection. Displayed alongside the average (center image) is the projection from the model in (B) which best fits the average (left image), compared to the projection from the model in (C) (right image). E, ribbon diagram of the Propeller/αA of CD11c/CD18 ectodomain crystal structure oriented as in Figure 2E, showing crystal contacts from two symmetry-related molecules (Calf-2 in gray and Thigh in magenta) that rotate MIDAS face of CD11cA domain by ∼35° (red arrows) relative to its position in the unrestricted EM model (colored as in Figure 2E). F, same Figure in (E) rotated clockwise by 135°.

Mentions: Because of the low resolution of our 3D EM map and the generally spherical nature of αA domain, we sought to independently assess its orientation relative to the Propeller domain by further examining the EM structure of a complex formed between the CD11b/CD18 ectodomain and Fab 107, a ligand mimetic that binds at the MIDAS face and stabilizes the integrin in the inactive state in the presence of the inhibitory Ca2+[16](Figure S1). The Fab fragment binds by an extensive and well-defined interface and thus allows identification of the CD11bA MIDAS face even at low resolution. Class averages generated from these particles (Figure 3A) closely resemble the averages generated from the ectodomain on its own, with the exception of a bilobed density attributable to the Fab. This indicates that the ectodomain within the complex is also in the bent conformation and that the Fab does not seem to interfere with the preferential orientation of the integrin on the grid. The presence of well-defined Fab density indicates that CD11bA/Fab107 complex possesses a limited range of orientations within the integrin, as a highly flexible link between CD11bA and the Propeller would result in the density for the former being averaged out. Attempts to generate a 3D map from the raw particles failed, with the resulting map not being interpretable as either integrin or Fab density. While this may be due to an underlying conformational heterogeneity, we think this unlikely due to the close correspondence between class averages generated from complexed and non-complexed particles. We rather attribute the different results for the two datasets to the different sizes of the masks used during the course of the reconstruction algorithm. The presence of the Fab required the use of a much larger mask than was needed for CD11b/CD18 ectodomain on its own to include all of the protein density. This necessarily included a larger area of the graphite background during the reconstruction, which combined with the low signal-to-noise ratio for these particles to defeat the 3D reconstruction algorithm. In support, a very tightly masked reconstruction of the ectodomain/Fab107 particles, which cuts off the Fab density resembles the map generated from CD11b/CD18 alone (not shown).


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)

EM analysis of the CD11b/CD18-Fab107 complex and orientation of the CD11bA MIDAS face.(A) Reference-free alignment and averaging of CD11b/CD18-Fab107 particles. The top row shows a k-means classification of 10,661 raw particles classified into 10 groups and averaged. The number of particles for each class is indicated at the top of each average. Several of the groups are similar, suggesting that there are fewer than 10 unique preferred orientations for the particles on the grid. The bottom row shows the model projection that best fits each of the particles shown in the top row. Projections in the bottom row were generated from the model shown in (B) which was generated by sequentially fitting a projection for the integrin followed by the projection for the CD11bA/Fab107 complex. For comparison, the model generated by fitting the CD11c/CD18 ectodomain X-ray structure is shown in (C). In this model, the CD11bA domain has the same relative orientation to the rest of the integrin as found in the X-ray structure. (D) Shows a direct comparison of the two models for the average, which most easily allows identification of individual integrin domains within the projection. Displayed alongside the average (center image) is the projection from the model in (B) which best fits the average (left image), compared to the projection from the model in (C) (right image). E, ribbon diagram of the Propeller/αA of CD11c/CD18 ectodomain crystal structure oriented as in Figure 2E, showing crystal contacts from two symmetry-related molecules (Calf-2 in gray and Thigh in magenta) that rotate MIDAS face of CD11cA domain by ∼35° (red arrows) relative to its position in the unrestricted EM model (colored as in Figure 2E). F, same Figure in (E) rotated clockwise by 135°.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585415&req=5

pone-0057951-g003: EM analysis of the CD11b/CD18-Fab107 complex and orientation of the CD11bA MIDAS face.(A) Reference-free alignment and averaging of CD11b/CD18-Fab107 particles. The top row shows a k-means classification of 10,661 raw particles classified into 10 groups and averaged. The number of particles for each class is indicated at the top of each average. Several of the groups are similar, suggesting that there are fewer than 10 unique preferred orientations for the particles on the grid. The bottom row shows the model projection that best fits each of the particles shown in the top row. Projections in the bottom row were generated from the model shown in (B) which was generated by sequentially fitting a projection for the integrin followed by the projection for the CD11bA/Fab107 complex. For comparison, the model generated by fitting the CD11c/CD18 ectodomain X-ray structure is shown in (C). In this model, the CD11bA domain has the same relative orientation to the rest of the integrin as found in the X-ray structure. (D) Shows a direct comparison of the two models for the average, which most easily allows identification of individual integrin domains within the projection. Displayed alongside the average (center image) is the projection from the model in (B) which best fits the average (left image), compared to the projection from the model in (C) (right image). E, ribbon diagram of the Propeller/αA of CD11c/CD18 ectodomain crystal structure oriented as in Figure 2E, showing crystal contacts from two symmetry-related molecules (Calf-2 in gray and Thigh in magenta) that rotate MIDAS face of CD11cA domain by ∼35° (red arrows) relative to its position in the unrestricted EM model (colored as in Figure 2E). F, same Figure in (E) rotated clockwise by 135°.
Mentions: Because of the low resolution of our 3D EM map and the generally spherical nature of αA domain, we sought to independently assess its orientation relative to the Propeller domain by further examining the EM structure of a complex formed between the CD11b/CD18 ectodomain and Fab 107, a ligand mimetic that binds at the MIDAS face and stabilizes the integrin in the inactive state in the presence of the inhibitory Ca2+[16](Figure S1). The Fab fragment binds by an extensive and well-defined interface and thus allows identification of the CD11bA MIDAS face even at low resolution. Class averages generated from these particles (Figure 3A) closely resemble the averages generated from the ectodomain on its own, with the exception of a bilobed density attributable to the Fab. This indicates that the ectodomain within the complex is also in the bent conformation and that the Fab does not seem to interfere with the preferential orientation of the integrin on the grid. The presence of well-defined Fab density indicates that CD11bA/Fab107 complex possesses a limited range of orientations within the integrin, as a highly flexible link between CD11bA and the Propeller would result in the density for the former being averaged out. Attempts to generate a 3D map from the raw particles failed, with the resulting map not being interpretable as either integrin or Fab density. While this may be due to an underlying conformational heterogeneity, we think this unlikely due to the close correspondence between class averages generated from complexed and non-complexed particles. We rather attribute the different results for the two datasets to the different sizes of the masks used during the course of the reconstruction algorithm. The presence of the Fab required the use of a much larger mask than was needed for CD11b/CD18 ectodomain on its own to include all of the protein density. This necessarily included a larger area of the graphite background during the reconstruction, which combined with the low signal-to-noise ratio for these particles to defeat the 3D reconstruction algorithm. In support, a very tightly masked reconstruction of the ectodomain/Fab107 particles, which cuts off the Fab density resembles the map generated from CD11b/CD18 alone (not shown).

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