Structural Insight into the Interaction between Platelet Integrin αIIbβ3 and Cytoskeletal Protein Skelemin

Lalit Deshmukh,Sergiy Tyukhtenko,Jianmin Liu,Joan E.B Fox,Jun Qin,Olga Vinogradova

doi:10.1074/jbc.m704666200

Lalit Deshmukh, Sergiy Tyukhtenko + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m704666200

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Abstract

Skelemin is a large cytoskeletal protein critical for cell morphology. Previous studies have suggested that its two-tandem immunoglobulin C2-like repeats (SkIgC4 and SkIgC5) are involved in binding to integrin beta3 cytoplasmic tail (CT), providing a mechanism for skelemin to regulate integrin-mediated signaling and cell spreading. Using NMR spectroscopy, we have studied the molecular details of the skelemin IgC45 interaction with the cytoplasmic face of integrin alphaIIbbeta3. Here, we show that skelemin IgC45 domains form a complex not only with integrin beta3 CT but also, surprisingly, with the integrin alphaIIb CT. Chemical shift mapping experiments demonstrate that both membrane-proximal regions of alphaIIb and beta3 CTs are involved in binding to skelemin. NMR structural determinations, combined with homology modeling, revealed that SkIgC4 and SkIgC5 both exhibited a conserved Ig-fold and both repeats were required for effective binding to and attenuation of alphaIIbbeta3 cytoplasmic complex. These data provide the first molecular insight into how skelemin may interact with integrins and regulate integrin-mediated signaling and cell spreading.

Highlights

The connection between extracellular matrix and the actin cytoskeleton is essential for cell extracellular matrix adhesion
Using NMR spectroscopy, we have studied the molecular details of the skelemin IgC45 interaction with the cytoplasmic face of integrin ␣IIb␤3
We show that skelemin immunoglobulin C2-like (IgC) repeats 4 and 5 exhibit a conserved Ig-fold, and we have characterized their interactions with integrin ␣IIb␤3 cytoplasmic tails

Summary

VanderWaal energy

TrNOE peaks do disappear but many remain. Because the ␣IIb CT binds to SkIgC45, some of the trNOE peaks could arise from this interaction. The additional peaks observed are consistent with our HSQC binding data (Fig. 2C) implicating the membrane-proximal region of the ␣IIb CT in the interaction. This latter spectral pattern is different from one of the ␣IIb CT in the presence of both MBP-␤3 CT and SkIgC45 (Fig. 3C). This observation suggests that SkIgC45 attenuates the ␣IIb␤3 CT clasp; complexity of the system and limits of the experimental approach do not allow us to conclude that this complex is fully disrupted by skelemin

DISCUSSION

Whether skelemin interacts with