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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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FLIM lifetimes of the Alexa488 donor fluorescence in the presence and absence of the acceptor FM.A) Representative Alexa488 fluorescence intensity and lifetime images. The panel shows examples of data collected in the absence (left, WT 488) and presence of the acceptor FM (right, WT 488+FM). The pseudocolor scale is shown at the bottom. B, histogram of measured Alexa488 lifetimes generated by integrating lifetime measurements for individual cells. The histogram bars show the number of cells in 50 ps bins for Alexa488 in the absence (blue) and presence (red) of the acceptor FM. The average and standard deviation were generated by fitting the histogram to a single Gaussian function (shown as a solid line).
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pone-0057951-g004: FLIM lifetimes of the Alexa488 donor fluorescence in the presence and absence of the acceptor FM.A) Representative Alexa488 fluorescence intensity and lifetime images. The panel shows examples of data collected in the absence (left, WT 488) and presence of the acceptor FM (right, WT 488+FM). The pseudocolor scale is shown at the bottom. B, histogram of measured Alexa488 lifetimes generated by integrating lifetime measurements for individual cells. The histogram bars show the number of cells in 50 ps bins for Alexa488 in the absence (blue) and presence (red) of the acceptor FM. The average and standard deviation were generated by fitting the histogram to a single Gaussian function (shown as a solid line).

Mentions: K562 cells stably expressing the recombinant integrin were stained with 20 µg/ml Fab107-Alexa488 at 4°C in a buffer containing 1 mM Ca2+ (Figure 4A, B). FLIM measurement in the absence of FM acceptor fluorophore produced a broad range of lifetimes of between 1,651 and 2,513 ps (Figure 4B). When these lifetimes were fit to a Gaussian distribution they were found to possess a mean of 2,234±13 ps (±95% confidence limits) with a standard deviation of 88±13 ps. This is similar to the 2,384 ps lifetime of unbound Fab-Alexa488 (not shown), suggesting that the Fab107-Alexa488 labeled cells are devoid of any endogenous fluorescence acceptor and are fully exposed to the solvent and not interacting with the protein, which might lengthen the lifetime. Solvent exposure indicates that the fluorophores, attached to the ε-amino group of the lysines, are able to tumble freely in solution and thus be randomly oriented. Only a minor population of cells seems to be intrinsically quenched. When the Fab107-Alexa488-labeled cells were further labeled with the membrane dye FM as an acceptor fluorophore, the Alexa488 fluorescence lifetime decreased significantly, generating a range of lifetimes between 1,354 and 2,099 ps. These lifetimes may also be fit to a single Gaussian with a mean of 1,529±14 ps and a standard deviation of 115±14 ps (Figure 4B). There is also a minor population of cells with lifetimes falling outside the fitted distribution, although in this case with longer lifetimes than the mean. In both cases the whole cell lifetimes may be fit to single Gaussian, with correlation coefficients of 0.83 and 0.88 for the quenched and unquenched measurements, respectively. Taking the mean values for the fits to the two datasets yields a FRET efficiency of ∼32% and a mean donor-acceptor separation distance (R) of ∼71Å, using the R0(2/3) value of 62Å for this donor-acceptor pair. Using the standard error of the mean produces an error estimate of only±∼1Å. Using±1σ for each of the fitted distributions produces a range of possible R values of between 76Å and 67Å. This result suggests that in the inactive conformation, the MIDAS face of CD11bA in full-length receptor lies in proximity to the cell's lipid bilayer, but not immediately adjacent.


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)

FLIM lifetimes of the Alexa488 donor fluorescence in the presence and absence of the acceptor FM.A) Representative Alexa488 fluorescence intensity and lifetime images. The panel shows examples of data collected in the absence (left, WT 488) and presence of the acceptor FM (right, WT 488+FM). The pseudocolor scale is shown at the bottom. B, histogram of measured Alexa488 lifetimes generated by integrating lifetime measurements for individual cells. The histogram bars show the number of cells in 50 ps bins for Alexa488 in the absence (blue) and presence (red) of the acceptor FM. The average and standard deviation were generated by fitting the histogram to a single Gaussian function (shown as a solid line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585415&req=5

pone-0057951-g004: FLIM lifetimes of the Alexa488 donor fluorescence in the presence and absence of the acceptor FM.A) Representative Alexa488 fluorescence intensity and lifetime images. The panel shows examples of data collected in the absence (left, WT 488) and presence of the acceptor FM (right, WT 488+FM). The pseudocolor scale is shown at the bottom. B, histogram of measured Alexa488 lifetimes generated by integrating lifetime measurements for individual cells. The histogram bars show the number of cells in 50 ps bins for Alexa488 in the absence (blue) and presence (red) of the acceptor FM. The average and standard deviation were generated by fitting the histogram to a single Gaussian function (shown as a solid line).
Mentions: K562 cells stably expressing the recombinant integrin were stained with 20 µg/ml Fab107-Alexa488 at 4°C in a buffer containing 1 mM Ca2+ (Figure 4A, B). FLIM measurement in the absence of FM acceptor fluorophore produced a broad range of lifetimes of between 1,651 and 2,513 ps (Figure 4B). When these lifetimes were fit to a Gaussian distribution they were found to possess a mean of 2,234±13 ps (±95% confidence limits) with a standard deviation of 88±13 ps. This is similar to the 2,384 ps lifetime of unbound Fab-Alexa488 (not shown), suggesting that the Fab107-Alexa488 labeled cells are devoid of any endogenous fluorescence acceptor and are fully exposed to the solvent and not interacting with the protein, which might lengthen the lifetime. Solvent exposure indicates that the fluorophores, attached to the ε-amino group of the lysines, are able to tumble freely in solution and thus be randomly oriented. Only a minor population of cells seems to be intrinsically quenched. When the Fab107-Alexa488-labeled cells were further labeled with the membrane dye FM as an acceptor fluorophore, the Alexa488 fluorescence lifetime decreased significantly, generating a range of lifetimes between 1,354 and 2,099 ps. These lifetimes may also be fit to a single Gaussian with a mean of 1,529±14 ps and a standard deviation of 115±14 ps (Figure 4B). There is also a minor population of cells with lifetimes falling outside the fitted distribution, although in this case with longer lifetimes than the mean. In both cases the whole cell lifetimes may be fit to single Gaussian, with correlation coefficients of 0.83 and 0.88 for the quenched and unquenched measurements, respectively. Taking the mean values for the fits to the two datasets yields a FRET efficiency of ∼32% and a mean donor-acceptor separation distance (R) of ∼71Å, using the R0(2/3) value of 62Å for this donor-acceptor pair. Using the standard error of the mean produces an error estimate of only±∼1Å. Using±1σ for each of the fitted distributions produces a range of possible R values of between 76Å and 67Å. This result suggests that in the inactive conformation, the MIDAS face of CD11bA in full-length receptor lies in proximity to the cell's lipid bilayer, but not immediately adjacent.

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