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Skelemin association with αIIbβ3 integrin: a structural model.

Gorbatyuk V, Nguyen K, Podolnikova NP, Deshmukh L, Lin X, Ugarova TP, Vinogradova O - Biochemistry (2014)

Bottom Line: Over the last two decades, our knowledge concerning intracellular events that regulate integrin's affinity to their soluble ligands has significantly improved.Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin αIIbβ3.This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut at Storrs , Storrs, Connecticut 06269, United States.

ABSTRACT
Over the last two decades, our knowledge concerning intracellular events that regulate integrin's affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal adhesions, still remains under-investigated. Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin αIIbβ3. The model of tertiary assembly is generated based upon NMR data and illuminates a potential link between the essential cell adhesion receptors and myosin filaments. This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.

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(A) ITC data for theSk45 interaction with αIIb fitted with a single bindingsite model. (B) The NMR ensemble of αIIb cytoplasmictail in the bound conformation as determined from trNOE data. Theconformer used for docking is shown in green. (C) A region of the 1H–15N HSQC spectra of Sk45 in absence (black)and presence of β3 (red) at the protein/peptide ratio1:3. Affected residues are labeled. (D) Effect of skelemin-derivedpeptide on αIIbβ3-expressing CHOcell adhesion. Microtiter wells were coated with 2.5 μg/mL offibrinogen and postcoated with 1% BSA. Calcein-labeled αIIbβ3-expressing CHO cells were incubatedwith different concentration of THIVWYKDEREISVDEKHD (•) orcontrol EREISAAAKHD (○) peptides for 20 min at 22 °C.After 30 min at 37 °C, nonadherent cells were removed, and adhesionwas determined. Data points are expressed as a percentage of controladhesion (in the absence of peptides) and are the mean of three individualexperiments performed with triplicate determinations in each experiment.
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fig4: (A) ITC data for theSk45 interaction with αIIb fitted with a single bindingsite model. (B) The NMR ensemble of αIIb cytoplasmictail in the bound conformation as determined from trNOE data. Theconformer used for docking is shown in green. (C) A region of the 1H–15N HSQC spectra of Sk45 in absence (black)and presence of β3 (red) at the protein/peptide ratio1:3. Affected residues are labeled. (D) Effect of skelemin-derivedpeptide on αIIbβ3-expressing CHOcell adhesion. Microtiter wells were coated with 2.5 μg/mL offibrinogen and postcoated with 1% BSA. Calcein-labeled αIIbβ3-expressing CHO cells were incubatedwith different concentration of THIVWYKDEREISVDEKHD (•) orcontrol EREISAAAKHD (○) peptides for 20 min at 22 °C.After 30 min at 37 °C, nonadherent cells were removed, and adhesionwas determined. Data points are expressed as a percentage of controladhesion (in the absence of peptides) and are the mean of three individualexperiments performed with triplicate determinations in each experiment.

Mentions: Previously, we defined skeleminbinding surface on platelet integrin αIIbβ3 and demonstrated that this interaction is consistent withan attenuated intersubunit clasp.11 Toaddress the mechanism of this interaction, and to define the thermodynamicforces driving the process, we have employed ITC. We used Sk4 andSk45 constructs titrated with either full-length αIIb cytoplasmic tail (Figure 4A, Figure S1.B) or short synthetic peptides correspondingto β3 N- or C-termini (FigureS1.A), as previously described.11 The results, summarized in Table 2, revealedvery weak interactions which were in the tens of micromolar range.These reactions were predominantly driven by entropy. For all cases,the stoichiometry of interactions was found to be one.


Skelemin association with αIIbβ3 integrin: a structural model.

Gorbatyuk V, Nguyen K, Podolnikova NP, Deshmukh L, Lin X, Ugarova TP, Vinogradova O - Biochemistry (2014)

(A) ITC data for theSk45 interaction with αIIb fitted with a single bindingsite model. (B) The NMR ensemble of αIIb cytoplasmictail in the bound conformation as determined from trNOE data. Theconformer used for docking is shown in green. (C) A region of the 1H–15N HSQC spectra of Sk45 in absence (black)and presence of β3 (red) at the protein/peptide ratio1:3. Affected residues are labeled. (D) Effect of skelemin-derivedpeptide on αIIbβ3-expressing CHOcell adhesion. Microtiter wells were coated with 2.5 μg/mL offibrinogen and postcoated with 1% BSA. Calcein-labeled αIIbβ3-expressing CHO cells were incubatedwith different concentration of THIVWYKDEREISVDEKHD (•) orcontrol EREISAAAKHD (○) peptides for 20 min at 22 °C.After 30 min at 37 °C, nonadherent cells were removed, and adhesionwas determined. Data points are expressed as a percentage of controladhesion (in the absence of peptides) and are the mean of three individualexperiments performed with triplicate determinations in each experiment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222533&req=5

fig4: (A) ITC data for theSk45 interaction with αIIb fitted with a single bindingsite model. (B) The NMR ensemble of αIIb cytoplasmictail in the bound conformation as determined from trNOE data. Theconformer used for docking is shown in green. (C) A region of the 1H–15N HSQC spectra of Sk45 in absence (black)and presence of β3 (red) at the protein/peptide ratio1:3. Affected residues are labeled. (D) Effect of skelemin-derivedpeptide on αIIbβ3-expressing CHOcell adhesion. Microtiter wells were coated with 2.5 μg/mL offibrinogen and postcoated with 1% BSA. Calcein-labeled αIIbβ3-expressing CHO cells were incubatedwith different concentration of THIVWYKDEREISVDEKHD (•) orcontrol EREISAAAKHD (○) peptides for 20 min at 22 °C.After 30 min at 37 °C, nonadherent cells were removed, and adhesionwas determined. Data points are expressed as a percentage of controladhesion (in the absence of peptides) and are the mean of three individualexperiments performed with triplicate determinations in each experiment.
Mentions: Previously, we defined skeleminbinding surface on platelet integrin αIIbβ3 and demonstrated that this interaction is consistent withan attenuated intersubunit clasp.11 Toaddress the mechanism of this interaction, and to define the thermodynamicforces driving the process, we have employed ITC. We used Sk4 andSk45 constructs titrated with either full-length αIIb cytoplasmic tail (Figure 4A, Figure S1.B) or short synthetic peptides correspondingto β3 N- or C-termini (FigureS1.A), as previously described.11 The results, summarized in Table 2, revealedvery weak interactions which were in the tens of micromolar range.These reactions were predominantly driven by entropy. For all cases,the stoichiometry of interactions was found to be one.

Bottom Line: Over the last two decades, our knowledge concerning intracellular events that regulate integrin's affinity to their soluble ligands has significantly improved.Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin αIIbβ3.This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut at Storrs , Storrs, Connecticut 06269, United States.

ABSTRACT
Over the last two decades, our knowledge concerning intracellular events that regulate integrin's affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal adhesions, still remains under-investigated. Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin αIIbβ3. The model of tertiary assembly is generated based upon NMR data and illuminates a potential link between the essential cell adhesion receptors and myosin filaments. This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.

Show MeSH
Related in: MedlinePlus