Three-dimensional Model of Human Platelet Integrin αIIbβ3 in Solution Obtained by Small Angle Neutron Scattering

Aurora Nogales,Carolina García,Javier Pérez,Phil Callow,Tiberio A Ezquerra,José González-Rodríguez

doi:10.1074/jbc.m109.050039

Abstract

Integrin alphaIIbbeta3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole alphaIIb beta3 in Ca(2+)/detergent solutions. Model projections after 90 degrees rotation along its long axis show an elongated and "arched" form (135 degrees) not observed before and a "handgun" form. This 20-nm-long structure is well defined, despite alphaIIb beta3 multidomain nature and expected segmental flexibility, with the largest region at the top, followed by two narrower and smaller regions at the bottom. Docking of this SANS envelope into the high resolution structure of alphaIIb beta3, reconstructed from crystallographic and NMR data, shows that the solution structure is less constrained, allows tentative assignment of the disposition of the alphaIIb and beta3 subunits and their domains within the model, and points out the structural analogies and differences of the SANS model with the crystallographic models of the recombinant ectodomains of alphaIIb beta3 and alphaV beta3 and with the cryo-electron microscopy model of whole alphaIIb beta3. The ectodomain is in the bent configuration at the top of the model, where alphaIIb and beta3 occupy the concave and convex sides, respectively, at the arched projection, with their bent knees at its apex. It follows the narrower transmembrane region and the cytoplasmic domains at the bottom end. AlphaIIb beta3 aggregated in Mn(2+)/detergent solutions, which impeded to get its SANS model.

Highlights

Previous x-ray crystallographic experiments showed that the recombinant ectodomains of integrin ␣V␤3 (r-␣V␤3 ectodomain) and ␣IIb␤3 (r-␣IIb␤3 ectodomain) and the complex of r-␣V␤3 ectodomain with an RGD peptide are in the bent configuration (10 –12)
When we measured the Triton X-100 bound to the protein eluted from the column and to the ultrafiltrated protein, we found that the protein solution coming directly from the column had the expected excess of Triton X-100 caused by the protein-bound Triton X-100 [20], that is, it was equilibrated with the column buffer, the ultrafiltrated protein solution had a larger excess because of a moderate concentration of the detergent micelles during ultrafiltration, which was not totally corrected by the extensive dialysis
This interpretation is supported by the values for both radius of gyration (Rg) (7.30 Ϯ 0.2 nm) and I(0) (6.48 ϫ 10Ϫ2 cmϪ1) obtained from the Guinier approximation in the qRg range of Ͻ1.3, which are too large compared with those obtained for the monomeric integrin in Ca2ϩ buffer and by the constantly increasing curvature up at low angle

Summary

Introduction

The linearity of the Guinier plot at the low q region [23] (Fig. 1a, inset) and the values of Rg and scattering intensity at zero angle I(0) derived from it, 5.16 Ϯ 0.04 nm and 4.67 ϫ 10Ϫ2 cmϪ1, respectively, indicate that ␣IIb␤3 in Ca2ϩ/detergent solutions was homogeneous and in monomer form.

Results

Conclusion