A. reduced, in which one or more disulfide bonds are broken. Furthermore, 2GPI can adopt multiple conformations (J-elongated, S-twisted, and O-circular). While strong evidence indicates that this 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 2GPI purified under native conditions is usually oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible HBEGF conformation, not circular or twisted, in which the N-terminal domain name I (DI) and the C-terminal domain name 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 this preferential binding of aPLs to phospholipid-bound 2GPI arises from the ability of its preexisting J-form to accumulate around the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of 2GPI provides a strategy to block pathogenic aPLs in APS. Keywords: beta-2 glycoprotein I, single-molecule biophysics, X-ray crystallography, structure-function, autoimmunity, thrombosis, antiphospholipid syndrome, complement system, coagulation, lipidCprotein conversation, structural biology, autoimmune disease, proteinCprotein conversation 2-Glycoprotein I (2GPI) is usually a 50-kDa multidomain glycoprotein that circulates in the plasma at a concentration of 0.2 mg/ml (1, 2) (Fig. 1and (14, 18,C20) and cause pregnancy complications resulting in fetal loss (21). Thus, a deeper understanding of the structural determinants of antigen-antibody recognition is likely to accelerate the development of new diagnostics and therapeutics for APS patients. An important feature of all aPLs, and especially highly pathogenic aPLs recognizing the epitope R39-R43 in the N-terminal domain PF6-AM name I of 2GPI, referred to here as anti-DI antibodies (22,C25), is usually that their detection requires proper immobilization of the antigen onto negatively charged surfaces or lipid membranes (26,C28), raising the question of whether the epitopes recognized by aPLs are cryptic in the circulating form of 2GPI. In support of this viewpoint, structural studies have documented that 2GPI can adopt alternative O-circular (29,C31), S-twisted (32), and J-elongated conformations (29,C31, 33, 34) featuring different exposures of DI and DV to the solvent (Fig. 11.15 M NaCl) and high pH (11.5) or in complex with the mAb 3B7 (29), bacterial lipopolysaccharide (LPS) labeled with gold nanoparticles (30), and protein H of (35), is believed to be the immunogenic conformation of the protein that interacts with aPLs, which appears when 2GPI binds to the membranes (Fig. 1and Fig. PF6-AM S1). The tag was then cleaved with enterokinase to generate the intact, mature protein (hr2GPI). Removal of the tag was confirmed by N-terminal sequencing (Fig. 2and Fig. S1). ST-2GPI was made to eliminate the enterokinase cleavage step, which was very laborious and not as PF6-AM efficient as expected. The presence of the short tag was confirmed by N-terminal sequencing and accounted for the different electrophoretic mobilities observed between recombinant and plasma purified protein before and after enzymatic removal of the N-glycosylations (Fig. 2LT-2GPI, hr2GPI, and ST-2GPI), highlighting the position and chemical composition of the N-terminal tag. The long tag (GGGS) were introduced to separate the three functional units of the tag to avoid the formation of secondary structure and ensure exposure of the tag to solvent. Removal of the LT with enterokinase generates hr2GPI. The short-tag version of 2GPI contains only the HPC4 purification tag (test. Results were considered significant at < 0.05 (*). To evaluate the functional integrity of the recombinant proteins, LT-2GPI, hr2GPI, and ST-2GPI were tested in several biochemical assays. 2GPI purified from human plasma using the perchloric acid method (p2GPI) was used as a control. Using surface plasmon resonance (SPR), a technique that allows us to measure association (on) and dissociation (off) rate constants in real time, we found that all variants interacted avidly with liposomes made up of negatively charged phospholipids, such as phosphatidylserine, yet they failed to interact with phospholipids made entirely of phosphatidylcholine (Fig. 2, and = 160.8, = 166.9, = 114.0= 159.3, = 173.2, = 115.2= 160.2, = 171.2, = 113.4????Molecules/asymmetric unit111????Resolution range (?)40C2.440C2.640C3.0????No. of observations513,296363,697158,386????No. of unique observations59,49748,50331,543????Completeness (%)98.8 (97.1)97.1 (77.1)98.8 (97.7)????bond lengths (?)0.0130.0100.011????RMSD angles (?)2.02.01.7????RMSD B (?2) (mm/ms/ss6V08, 6V06 6V09, and 6V08 6V09, respectively) and also very similar to the published ones (RMSD of 0.810 ?) (33, 34) (Fig. 42.4 and 2.6 ?) (Fig. 4and and and 0.145 1 m, Fig. 57.4 10.0, Fig. 5linear globular, and, similar to smFRET, therefore is usually ideal to detect large conformational changes in 2GPI. The radius of gyration ((160 ?), agree very well with the values calculated using the J-elongated structure captured by X-ray crystallography (= 45.22 and from the SAXS data of ST-2GPI returned a linear.