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The interaction of ß₂-glycoprotein I domain V with chaperonin GroEL: The similarity with the domain V and membrane interaction

¹ Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan
² National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565, Japan

Reprint requests to: Yuji Goto, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan; e-mail: ygoto{at}protein.osaka-u.ac.jp; fax: 81-6-6879-8616.

To clarify the mechanism of interaction between chaperonin GroEL and substrate proteins, we studied the conformational changes; of the fifth domain of human ß₂-glycoprotein I upon binding to GroEL. The fifth domain has a large flexible loop, containing several hydrophobic residues surrounded by positively charged residues, which has been proposed to be responsible for the binding of ß₂-glycoprotein I to negatively charged phospholipid membranes. The reduction by dithiothreitol of the three intramolecular disulfide bonds of the fifth domain was accelerated in the presence of stoichiometric amounts of GroEL, indicating that the fifth domain was destabilized upon interaction with GroEL. To clarify the GroEL-induced destabilization at the atomic level, we performed H/²H exchange of amide protons using heteronuclear NMR spectroscopy. The presence of GroEL promoted the H/²H exchange of most of the protected amide protons, suggesting that, although the flexible loop of the fifth domain is likely to be responsible for the initiation of binding to GroEL, the interaction with GroEL destabilizes the overall conformation of the fifth domain.

Keywords: ß2-Glycoprotein I; chaperonin GroEL; disulfide bond reduction; heteronuclear NMR; H/2H exchange

Abbreviations: ß2GPI, ß2-glycoprotein I • Domain V, recombinant fifth domain of ß2GPI • DTT, dithiothreitol • HSQC, heteronuclear single quantum coherence • NMR, nuclear magnetic resonance • PF, protection factor • p2Hr, pH meter reading of 2H2O solution • HPLC, high-performance liquid chromatography

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