Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Dec 28;107(52):22481-6.
doi: 10.1073/pnas.1015545107. Epub 2010 Dec 14.

NMR Analysis of the alphaIIb beta3 Cytoplasmic Interaction Suggests a Mechanism for Integrin Regulation

Affiliations
Free PMC article

NMR Analysis of the alphaIIb beta3 Cytoplasmic Interaction Suggests a Mechanism for Integrin Regulation

Douglas G Metcalf et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The integrin αIIbβ3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbβ3, the TM and cytoplasmic domains of αIIb and β3 form a heterodimer that constrains αIIbβ3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogen-deuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the β3 cytoplasmic domain. The β3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the β3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the β3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
A disulfide-linked construct for the αIIbβ3 cytoplasmic domain heterodimer. (A) Cartoon depicting the position of the engineered disulfide bond between αIIb Met987Cys and β3 Leu712Cys and its relation to protein binding sites in the αIIb and β3 cytoplasmic domains. (B) Sequence of the disulfide-linked construct. (C) Cα chemical shifts define three helical regions of β3 in the disulfide-linked heterodimer. Differences between the Cα resonances in the αIIbβ3 heterodimer and β3 monomer are shown as bars above the graph. (D) Side chain chemical shifts of aliphatic atoms, visualized in the 13C HSQC spectrum, are sensitive to protein interactions. The blue and purple signals correspond to aliphatic atoms in the disulfide-linked αIIbβ3 heterodimer and the red and green overlay corresponds to those in the αIIb and β3 monomers. (Purple and green signal is folded in the carbon dimension by 20 ppm.)
Fig. 2.
Fig. 2.
Structure of the β3 cytoplasmic domain. (A) Twenty structures in the NMR ensemble, consisting of two regions encompassing residues Leu713-Trp739 and Asp740-Thr762, were aligned separately over well-ordered regions. Domains interacting with αIIb, talin, kindlin-3, and Src kinase are enclosed by dashed lines, and side chains that make up interfaces are depicted as sticks. The two β3 NPXY motifs are colored magenta and the Src-binding RGT motif is colored cyan. (B) A single-residue hinge at Arg724 is stabilized by packing between residues Ile721 and Phe727 (green) and an electrostatic interaction between Arg724 (blue) and Glu726 (red). (C) The first cytosolic helix (residues Glu726-Arg736) is stabilized by a network of complementary charged residues. Positively charged residues are colored blue and negatively charged residues red.
Fig. 3.
Fig. 3.
Backbone dynamics of the αIIbβ3 cytoplasmic domain heterodimer. (A) Backbone dynamics were inferred from intraamide 1H-15N HSQC peak heights (14) and were compared with the corresponding isolated β3 construct (14). (B) 15N HSQC spectrum of the disulfide-linked complex before and after HDX. The β3 backbone amides Ile714-Ile721 are protected from HDX, suggesting they form stable hydrogen bonds in a helix. Other helical regions exchange with deuterium on the minute time scale, suggesting they make excursions from helical conformations.
Fig. 4.
Fig. 4.
HDX of the β3 cytoplasmic domain conjugated to lipid. After incubating β3 cytoplasmic domain peptides, either covalently tethered to phospholipid bilayers or free in solution, in D2O for various periods of time, deuterium exchange was quenched at pH 2.5, and the peptides were injected onto a pepsin column in tandem with LC-MS to resolve fragments corresponding to residues Glu733-Thr753 (A), Asn743-Thr762 (B), and Asn756-Thr762 (C). Asn743-Thr762 was not observed in the absence the bilayers (B). The black line is a theoretical curve based on the intrinsic peptide exchange rate. The green line is HDX of lipid-tethered β3, the red line is HDX of free β3. Residues highlighted in light blue are expected to be protected by intrahelical hydrogen bonding. Residues highlighted in orange are either unstructured or at the N termini of their respective helices.
Fig. 5.
Fig. 5.
A linked equilibrium describing the activation of αIIbβ3 by intracellular signaling molecules. The β3 cytoplasmic tail exists in equilibrium between conformations that interact with the membrane and conformations exposed to the cytoplasm. To the left is a resting conformation with helices CytoH1 and CytoH2 interacting with the bilayer. The fully inactive state is not competent to bind talin or kindlin because the membrane obstructs access to β3. [(Inset A) Key hydrophobic side chains, shown in green, are necessary for interactions with talin and kindlin and are obscured by interactions with the bilayer.] To the right is a fully active conformation with separated TM and cytoplasmic domains. In between exists an ensemble of states in which the integrin is not active, but the β3 cytoplasmic domain samples a wide range of conformations and is accessible to intracellular molecules. [(Inset B) The dynamic nature of the β3 cytoplasmic domain makes protein–protein interaction motifs accessible. Dissociation of αIIb and extension of the β3 TM-proximal helix to join CytoH1 allows talin to bind and places positively charged residues on talin’s upper surface in a favorable position to interact electrostatically with the inner membrane leaflet. β3 is also now available to bind kindlin.] Although talin and kindlin can bind the ensemble of free cytoplasmic domains in the middle column, available data are insufficient to predict the order of binding. Therefore, equilibria depicting talin binding first (top) and kindlin binding first (bottom) are shown. However the fact that both kindlin and talin are necessary for activation suggests that both bind to the integrin cytoplasmic domain.

Similar articles

See all similar articles

Cited by 33 articles

See all "Cited by" articles

Publication types

MeSH terms

Associated data

LinkOut - more resources

Feedback