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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 refinement of the CD11b/CD18 ectodomain.(A) Example images from the final round of refinement. On the left is an average image generated from reference-free alignment and averaging which preceded the refinement itself. The averages have been displayed with the final map projection that they most closely resemble. Results from the final round of refinement are displayed adjacent to the reference-free average, showing from left to right of each panel the average from reference free alignment (number 1), an example raw particle from the final projection class (number 2), the average generated from the final projection class (number 3), and the projection from the final map for this class (number 4). The letters on the left indicate the class in the distribution histogram (B). The side of each box is 232Å. (B) Projection histogram showing the number of particles identified as belonging to a particular Euler angle orientation for the final round of refinement. Each circle indicates a particular class while the number of particles is indicated as a gray scale. The scale bar is indicated to the right. The letters refer to the classes shown in (A). (C) Fourier shell correlation analysis to determine the resolution of the final map. The total dataset was separated randomly into two equal groups. The particle classes for each group were independently aligned with the final map projection and averaged to generate two independent maps. The graph displays the correlation in Fourier space between the maps. The resolution of 1/(26 Å) is determined by a value of 50% correlation.
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pone-0057951-g001: EM refinement of the CD11b/CD18 ectodomain.(A) Example images from the final round of refinement. On the left is an average image generated from reference-free alignment and averaging which preceded the refinement itself. The averages have been displayed with the final map projection that they most closely resemble. Results from the final round of refinement are displayed adjacent to the reference-free average, showing from left to right of each panel the average from reference free alignment (number 1), an example raw particle from the final projection class (number 2), the average generated from the final projection class (number 3), and the projection from the final map for this class (number 4). The letters on the left indicate the class in the distribution histogram (B). The side of each box is 232Å. (B) Projection histogram showing the number of particles identified as belonging to a particular Euler angle orientation for the final round of refinement. Each circle indicates a particular class while the number of particles is indicated as a gray scale. The scale bar is indicated to the right. The letters refer to the classes shown in (A). (C) Fourier shell correlation analysis to determine the resolution of the final map. The total dataset was separated randomly into two equal groups. The particle classes for each group were independently aligned with the final map projection and averaged to generate two independent maps. The graph displays the correlation in Fourier space between the maps. The resolution of 1/(26 Å) is determined by a value of 50% correlation.

Mentions: We determined the 3D EM structure of CD11b/CD18 ectodomain in buffer containing the inhibitory Ca2+[24], which stabilizes the inactive state. The full dataset contained 8,825 particles selected from negatively stained micrographs using an automated, model-free algorithm. Preliminary analysis was conducted with reference-free alignment and classification, where raw particle images are aligned to a common center and classified using features extracted from the covariance of the global average. This method, which does not rely on any presupposition of the protein's structure, produced several averages, which closely resemble views expected from a bent conformation of the ectodomain (Figure 1). Indeed, all of the resulting averages were compact, and the majority could be assigned to a bent conformation. Side views displayed clear density for CD11bA domain, indicating that in CD11b this domain is relatively stable, as conformational flexibility would have led to its density being averaged out.


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 refinement of the CD11b/CD18 ectodomain.(A) Example images from the final round of refinement. On the left is an average image generated from reference-free alignment and averaging which preceded the refinement itself. The averages have been displayed with the final map projection that they most closely resemble. Results from the final round of refinement are displayed adjacent to the reference-free average, showing from left to right of each panel the average from reference free alignment (number 1), an example raw particle from the final projection class (number 2), the average generated from the final projection class (number 3), and the projection from the final map for this class (number 4). The letters on the left indicate the class in the distribution histogram (B). The side of each box is 232Å. (B) Projection histogram showing the number of particles identified as belonging to a particular Euler angle orientation for the final round of refinement. Each circle indicates a particular class while the number of particles is indicated as a gray scale. The scale bar is indicated to the right. The letters refer to the classes shown in (A). (C) Fourier shell correlation analysis to determine the resolution of the final map. The total dataset was separated randomly into two equal groups. The particle classes for each group were independently aligned with the final map projection and averaged to generate two independent maps. The graph displays the correlation in Fourier space between the maps. The resolution of 1/(26 Å) is determined by a value of 50% correlation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585415&req=5

pone-0057951-g001: EM refinement of the CD11b/CD18 ectodomain.(A) Example images from the final round of refinement. On the left is an average image generated from reference-free alignment and averaging which preceded the refinement itself. The averages have been displayed with the final map projection that they most closely resemble. Results from the final round of refinement are displayed adjacent to the reference-free average, showing from left to right of each panel the average from reference free alignment (number 1), an example raw particle from the final projection class (number 2), the average generated from the final projection class (number 3), and the projection from the final map for this class (number 4). The letters on the left indicate the class in the distribution histogram (B). The side of each box is 232Å. (B) Projection histogram showing the number of particles identified as belonging to a particular Euler angle orientation for the final round of refinement. Each circle indicates a particular class while the number of particles is indicated as a gray scale. The scale bar is indicated to the right. The letters refer to the classes shown in (A). (C) Fourier shell correlation analysis to determine the resolution of the final map. The total dataset was separated randomly into two equal groups. The particle classes for each group were independently aligned with the final map projection and averaged to generate two independent maps. The graph displays the correlation in Fourier space between the maps. The resolution of 1/(26 Å) is determined by a value of 50% correlation.
Mentions: We determined the 3D EM structure of CD11b/CD18 ectodomain in buffer containing the inhibitory Ca2+[24], which stabilizes the inactive state. The full dataset contained 8,825 particles selected from negatively stained micrographs using an automated, model-free algorithm. Preliminary analysis was conducted with reference-free alignment and classification, where raw particle images are aligned to a common center and classified using features extracted from the covariance of the global average. This method, which does not rely on any presupposition of the protein's structure, produced several averages, which closely resemble views expected from a bent conformation of the ectodomain (Figure 1). Indeed, all of the resulting averages were compact, and the majority could be assigned to a bent conformation. Side views displayed clear density for CD11bA domain, indicating that in CD11b this domain is relatively stable, as conformational flexibility would have led to its density being averaged out.

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