The J-elongated conformation of β2-glycoprotein I predominates in solution: implications for our understanding of antiphospholipid syndrome

Eliza Ruben,William Planer,Mathivanan Chinnaraj,Zhiwei Chen,Xiaobing Zuo,Vittorio Pengo,Vincenzo De Filippis,Ravi K Alluri,Keith R Mccrae,Paolo Macor,Francesco Tedesco,Nicola Pozzi

doi:10.1074/jbc.ra120.013939

Abstract

β2-Glycoprotein I (β2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound β2GPI to that in solution. β2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, β2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant β2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound β2GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of β2GPI provides a strategy to block pathogenic aPLs in APS.

Highlights

182–185), and Lnk4 [3]
Forgotten for many years since its discovery in 1961 [1], b2-Glycoprotein I (b2GPI) gained popularity in the fields of hematology and rheumatology in 1990, when it was recognized by two independent studies as the dominant antigen of antiphospholipid antibodies in antiphospholipid syndrome (APS) [11,12,13], a lifethreatening blood-clotting disorder characterized by vascular thrombosis and pregnancy morbidity [10, 14]
In support of this viewpoint, structural studies have documented that b2GPI can adopt alternative O-circular [29,30,31], S-twisted [32], and J-elongated conformations [29,30,31, 33, 34] featuring different exposures of domain I (DI) and domain V (DV) to the solvent (Fig. 1B) and have led to the proposition of a model whereby the O-circular form of b2GPI, which was captured by negative-stain EM [29] and atomic force microscopy (AFM) [31] using b2GPI purified from plasma using mild conditions, is the most abundant (.90%) protein conformation of b2GPI under physiological conditions, which is immunologically inert and incapable of reacting with antiphospholipid antibodies (aPLs) (Fig. 1B, left)

Summary

Introduction

182–185), and Lnk4 (residues 242–244) [3]. Domains I–IV are canonical complement control protein (CCP) domains, each containing 4 cysteine residues typically forming 2 disulfide bonds [4, 5]. ST-b2GPI, for which extra electron density was observed at the N terminus (Fig. 3D), crystallized under similar conditions and in the same space group as hrb2GPI and pb2GPI, confirming minimal structural perturbation introduced by the artificial tag.

Results

Conclusion