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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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Modeling of the relative membrane orientation of CD11b/CD18 by combining the FLIM and EM data.A) A display of the allowed orientations of the integrin ectodomain. To determine the allowed orientations, the protein is first rotated in the membrane plane and subsequently rotated down. For each rotation pair, the model is evaluated for clashes with the membrane. Shown in magenta are the centroids for Fab107 for all of the resulting angles. For illustration purposes, the integrin α- (blue), β- (red) subunits and Fab107 (green) chains are displayed as a ribbon diagram for one of the resulting orientations. A section of a model DMPC membrane is shown as a wire diagram. The model of the CD11b/CD18 ectodomain is taken from the fit to the EM map. The distance of the centroid for the displayed model is compatible with the measured FLIM distance. B) The same view as in (A) but rotated in the y-axis by 45°. The α/β TM domains (modeled after the NMR structure of αIIbβ3 TM domains [46]) are displayed for illustrative purposes only, and were not used in the orientation search.
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pone-0057951-g005: Modeling of the relative membrane orientation of CD11b/CD18 by combining the FLIM and EM data.A) A display of the allowed orientations of the integrin ectodomain. To determine the allowed orientations, the protein is first rotated in the membrane plane and subsequently rotated down. For each rotation pair, the model is evaluated for clashes with the membrane. Shown in magenta are the centroids for Fab107 for all of the resulting angles. For illustration purposes, the integrin α- (blue), β- (red) subunits and Fab107 (green) chains are displayed as a ribbon diagram for one of the resulting orientations. A section of a model DMPC membrane is shown as a wire diagram. The model of the CD11b/CD18 ectodomain is taken from the fit to the EM map. The distance of the centroid for the displayed model is compatible with the measured FLIM distance. B) The same view as in (A) but rotated in the y-axis by 45°. The α/β TM domains (modeled after the NMR structure of αIIbβ3 TM domains [46]) are displayed for illustrative purposes only, and were not used in the orientation search.

Mentions: We analyzed our FLIM data by systematically evaluating atomic models to determine the relative orientation of the integrin ectodomain to the membrane plane. The lipid bilayer is modeled as a plane (see methods), with the FLIM distance taken as the shortest distance from the centroid to any point on the plane. Orientation of the integrin on the lipid bilayer requires specifying two angles, the angle between the long axis of Calf-1/Calf-2 domains and the membrane plane (the tilt angle, Θ, being 0° or 90° when the long axis of Calf-2 is either flush with or perpendicular to the membrane, respectively) and the direction of the tilt angle (Φ). With only a single FLIM distance measurement, a large number of combinations of the two angles may be found. Our measured mean FLIM distance of 71Å between Fab107 and the lipid headgroups provided the initial constraint. To determine the angles that satisfy this distance, an atomic model of the integrin/Fab107 complex generated by the fit to the EM data (Figure 3B) is rotated along all available angles and the resulting distance between the Fab107 centroid and the membrane plane measured at each point. There is not as yet an X-ray structure of any intact integrin, and this has compelled our modeling to be performed with ectodomain structures instead, all of which adopt similar bent conformations, where the legs are bent back to position the membrane junctional regions proximal to the head domain. Current structural investigations on full-length integrins lack atomic (or even near-atomic) resolution but do agree that the full-length structure of the inactive conformation is also compact, with its ectodomain likely bent [28], [29]. As a result, our ectodomain model was rotated through a full 360° in Φ and 180° in Θ, which allows the leg domains to vary from completely vertical to lying flat on the surface of the membrane to being buried vertically in the membrane. To check orientations for clashes with the membrane, the membrane was modeled as a simple plane in x-y, and excluding models where any atom dipped below it. A 5° search for both angles generated a total of 114 possible orientations where the CD11b/CD18-Fab107 complex would not clash with the membrane (Figure 5A,B). Applying the further FLIM distance constraint of 71Å between Fab107 and membrane plane further reduced the number of orientations to eight. These solutions possess a range of tilt angles (Θ) of 50° to 60° and an average angle of 52.5°. To check our ectodomain model, we considered the CD11c/CD18 X-ray structure, which does not fit well with our EM ectodomain structure but which might represent the intact full-length structure. This latter model, generated by aligning the αA domains in the CD11c/CD18 and CD11bA/Fab107 X-ray structures, failed to produce any orientation that simultaneously satisfied the membrane clash and FLIM distance criteria.


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)

Modeling of the relative membrane orientation of CD11b/CD18 by combining the FLIM and EM data.A) A display of the allowed orientations of the integrin ectodomain. To determine the allowed orientations, the protein is first rotated in the membrane plane and subsequently rotated down. For each rotation pair, the model is evaluated for clashes with the membrane. Shown in magenta are the centroids for Fab107 for all of the resulting angles. For illustration purposes, the integrin α- (blue), β- (red) subunits and Fab107 (green) chains are displayed as a ribbon diagram for one of the resulting orientations. A section of a model DMPC membrane is shown as a wire diagram. The model of the CD11b/CD18 ectodomain is taken from the fit to the EM map. The distance of the centroid for the displayed model is compatible with the measured FLIM distance. B) The same view as in (A) but rotated in the y-axis by 45°. The α/β TM domains (modeled after the NMR structure of αIIbβ3 TM domains [46]) are displayed for illustrative purposes only, and were not used in the orientation search.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585415&req=5

pone-0057951-g005: Modeling of the relative membrane orientation of CD11b/CD18 by combining the FLIM and EM data.A) A display of the allowed orientations of the integrin ectodomain. To determine the allowed orientations, the protein is first rotated in the membrane plane and subsequently rotated down. For each rotation pair, the model is evaluated for clashes with the membrane. Shown in magenta are the centroids for Fab107 for all of the resulting angles. For illustration purposes, the integrin α- (blue), β- (red) subunits and Fab107 (green) chains are displayed as a ribbon diagram for one of the resulting orientations. A section of a model DMPC membrane is shown as a wire diagram. The model of the CD11b/CD18 ectodomain is taken from the fit to the EM map. The distance of the centroid for the displayed model is compatible with the measured FLIM distance. B) The same view as in (A) but rotated in the y-axis by 45°. The α/β TM domains (modeled after the NMR structure of αIIbβ3 TM domains [46]) are displayed for illustrative purposes only, and were not used in the orientation search.
Mentions: We analyzed our FLIM data by systematically evaluating atomic models to determine the relative orientation of the integrin ectodomain to the membrane plane. The lipid bilayer is modeled as a plane (see methods), with the FLIM distance taken as the shortest distance from the centroid to any point on the plane. Orientation of the integrin on the lipid bilayer requires specifying two angles, the angle between the long axis of Calf-1/Calf-2 domains and the membrane plane (the tilt angle, Θ, being 0° or 90° when the long axis of Calf-2 is either flush with or perpendicular to the membrane, respectively) and the direction of the tilt angle (Φ). With only a single FLIM distance measurement, a large number of combinations of the two angles may be found. Our measured mean FLIM distance of 71Å between Fab107 and the lipid headgroups provided the initial constraint. To determine the angles that satisfy this distance, an atomic model of the integrin/Fab107 complex generated by the fit to the EM data (Figure 3B) is rotated along all available angles and the resulting distance between the Fab107 centroid and the membrane plane measured at each point. There is not as yet an X-ray structure of any intact integrin, and this has compelled our modeling to be performed with ectodomain structures instead, all of which adopt similar bent conformations, where the legs are bent back to position the membrane junctional regions proximal to the head domain. Current structural investigations on full-length integrins lack atomic (or even near-atomic) resolution but do agree that the full-length structure of the inactive conformation is also compact, with its ectodomain likely bent [28], [29]. As a result, our ectodomain model was rotated through a full 360° in Φ and 180° in Θ, which allows the leg domains to vary from completely vertical to lying flat on the surface of the membrane to being buried vertically in the membrane. To check orientations for clashes with the membrane, the membrane was modeled as a simple plane in x-y, and excluding models where any atom dipped below it. A 5° search for both angles generated a total of 114 possible orientations where the CD11b/CD18-Fab107 complex would not clash with the membrane (Figure 5A,B). Applying the further FLIM distance constraint of 71Å between Fab107 and membrane plane further reduced the number of orientations to eight. These solutions possess a range of tilt angles (Θ) of 50° to 60° and an average angle of 52.5°. To check our ectodomain model, we considered the CD11c/CD18 X-ray structure, which does not fit well with our EM ectodomain structure but which might represent the intact full-length structure. This latter model, generated by aligning the αA domains in the CD11c/CD18 and CD11bA/Fab107 X-ray structures, failed to produce any orientation that simultaneously satisfied the membrane clash and FLIM distance criteria.

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