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Real-time analysis of conformation-sensitive antibody binding provides new insights into integrin conformational regulation.

Chigaev A, Waller A, Amit O, Halip L, Bologa CG, Sklar LA - J. Biol. Chem. (2009)

Bottom Line: We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope.Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding.Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA. achigaev@salud.unm.edu

ABSTRACT
Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of alpha(4)beta(1) integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC(50) for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

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Related in: MedlinePlus

Model of integrin conformations. Three-dimensional structures for VLA-4 multiple conformational states have been generated as described under “Experimental Procedures” by combining the integrin structural information existent in Protein Data Bank and relevant literature data (21, 22). The integrin head is colored in red, the “upper legs” are in blue, and the “lower legs” are in green. In the model Ser370, Glu371, and Lys417, which represent HUTS epitope (34), are shown by purple space fill. The VLA-4 bent closed conformation is modeled based on crystal structure of αVβ3 integrin (structure A). Structure A represents a bent low affinity state (resting state), having the HUTS-21 epitope unexposed (purple spheres). As in the template, the N termini of α and β subunits are set into an ovoid-like arrangement from which two parallel tails come out. Because the crystal structure does not offer any structural information regarding EGF-domains, no EGF domains are shown on A and A′ (see the “Experimental Procedures” for details). The conformational change induced by the occupancy of the ligand pocket (structure B) was modeled in two steps. First, the β1 structure built based on the β3 open headpiece was translated into the structure A coordinate system. By overlapping the 1L5G (closed bent conformation) and 1TXV (open conformation) structures, we found that the distance between C termini domains of α and β subunits is ∼15 Å, and the distance between α and β “knees” is ∼70 Å. These constraints were used to build the bent open conformation of VLA-4 (structure B). This structure has the outward swing of the hybrid domain, which causes the exposure of HUTS-21 epitope and represents the low affinity state of the integrin. The unbent conformation with closed and open headpiece (structure C and D, respectively) have been obtained by adjusting the torsion angles at the knees of α4 and β1 subunits in A and B structures. In these operations the upper and lower legs of each subunit were considered as two rigid systems. All final conformations have been minimized with the Biopolymer module from Sybyl (SYBYL 7.3, Tripos International).
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fig6: Model of integrin conformations. Three-dimensional structures for VLA-4 multiple conformational states have been generated as described under “Experimental Procedures” by combining the integrin structural information existent in Protein Data Bank and relevant literature data (21, 22). The integrin head is colored in red, the “upper legs” are in blue, and the “lower legs” are in green. In the model Ser370, Glu371, and Lys417, which represent HUTS epitope (34), are shown by purple space fill. The VLA-4 bent closed conformation is modeled based on crystal structure of αVβ3 integrin (structure A). Structure A represents a bent low affinity state (resting state), having the HUTS-21 epitope unexposed (purple spheres). As in the template, the N termini of α and β subunits are set into an ovoid-like arrangement from which two parallel tails come out. Because the crystal structure does not offer any structural information regarding EGF-domains, no EGF domains are shown on A and A′ (see the “Experimental Procedures” for details). The conformational change induced by the occupancy of the ligand pocket (structure B) was modeled in two steps. First, the β1 structure built based on the β3 open headpiece was translated into the structure A coordinate system. By overlapping the 1L5G (closed bent conformation) and 1TXV (open conformation) structures, we found that the distance between C termini domains of α and β subunits is ∼15 Å, and the distance between α and β “knees” is ∼70 Å. These constraints were used to build the bent open conformation of VLA-4 (structure B). This structure has the outward swing of the hybrid domain, which causes the exposure of HUTS-21 epitope and represents the low affinity state of the integrin. The unbent conformation with closed and open headpiece (structure C and D, respectively) have been obtained by adjusting the torsion angles at the knees of α4 and β1 subunits in A and B structures. In these operations the upper and lower legs of each subunit were considered as two rigid systems. All final conformations have been minimized with the Biopolymer module from Sybyl (SYBYL 7.3, Tripos International).

Mentions: The on-line version of this article (available at http://www.jbc.org) contains supplemental structures shown in Fig. 6.


Real-time analysis of conformation-sensitive antibody binding provides new insights into integrin conformational regulation.

Chigaev A, Waller A, Amit O, Halip L, Bologa CG, Sklar LA - J. Biol. Chem. (2009)

Model of integrin conformations. Three-dimensional structures for VLA-4 multiple conformational states have been generated as described under “Experimental Procedures” by combining the integrin structural information existent in Protein Data Bank and relevant literature data (21, 22). The integrin head is colored in red, the “upper legs” are in blue, and the “lower legs” are in green. In the model Ser370, Glu371, and Lys417, which represent HUTS epitope (34), are shown by purple space fill. The VLA-4 bent closed conformation is modeled based on crystal structure of αVβ3 integrin (structure A). Structure A represents a bent low affinity state (resting state), having the HUTS-21 epitope unexposed (purple spheres). As in the template, the N termini of α and β subunits are set into an ovoid-like arrangement from which two parallel tails come out. Because the crystal structure does not offer any structural information regarding EGF-domains, no EGF domains are shown on A and A′ (see the “Experimental Procedures” for details). The conformational change induced by the occupancy of the ligand pocket (structure B) was modeled in two steps. First, the β1 structure built based on the β3 open headpiece was translated into the structure A coordinate system. By overlapping the 1L5G (closed bent conformation) and 1TXV (open conformation) structures, we found that the distance between C termini domains of α and β subunits is ∼15 Å, and the distance between α and β “knees” is ∼70 Å. These constraints were used to build the bent open conformation of VLA-4 (structure B). This structure has the outward swing of the hybrid domain, which causes the exposure of HUTS-21 epitope and represents the low affinity state of the integrin. The unbent conformation with closed and open headpiece (structure C and D, respectively) have been obtained by adjusting the torsion angles at the knees of α4 and β1 subunits in A and B structures. In these operations the upper and lower legs of each subunit were considered as two rigid systems. All final conformations have been minimized with the Biopolymer module from Sybyl (SYBYL 7.3, Tripos International).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682882&req=5

fig6: Model of integrin conformations. Three-dimensional structures for VLA-4 multiple conformational states have been generated as described under “Experimental Procedures” by combining the integrin structural information existent in Protein Data Bank and relevant literature data (21, 22). The integrin head is colored in red, the “upper legs” are in blue, and the “lower legs” are in green. In the model Ser370, Glu371, and Lys417, which represent HUTS epitope (34), are shown by purple space fill. The VLA-4 bent closed conformation is modeled based on crystal structure of αVβ3 integrin (structure A). Structure A represents a bent low affinity state (resting state), having the HUTS-21 epitope unexposed (purple spheres). As in the template, the N termini of α and β subunits are set into an ovoid-like arrangement from which two parallel tails come out. Because the crystal structure does not offer any structural information regarding EGF-domains, no EGF domains are shown on A and A′ (see the “Experimental Procedures” for details). The conformational change induced by the occupancy of the ligand pocket (structure B) was modeled in two steps. First, the β1 structure built based on the β3 open headpiece was translated into the structure A coordinate system. By overlapping the 1L5G (closed bent conformation) and 1TXV (open conformation) structures, we found that the distance between C termini domains of α and β subunits is ∼15 Å, and the distance between α and β “knees” is ∼70 Å. These constraints were used to build the bent open conformation of VLA-4 (structure B). This structure has the outward swing of the hybrid domain, which causes the exposure of HUTS-21 epitope and represents the low affinity state of the integrin. The unbent conformation with closed and open headpiece (structure C and D, respectively) have been obtained by adjusting the torsion angles at the knees of α4 and β1 subunits in A and B structures. In these operations the upper and lower legs of each subunit were considered as two rigid systems. All final conformations have been minimized with the Biopolymer module from Sybyl (SYBYL 7.3, Tripos International).
Mentions: The on-line version of this article (available at http://www.jbc.org) contains supplemental structures shown in Fig. 6.

Bottom Line: We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope.Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding.Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA. achigaev@salud.unm.edu

ABSTRACT
Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of alpha(4)beta(1) integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC(50) for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

Show MeSH
Related in: MedlinePlus