Thioredoxin fold as homodimerization module in the putative chaperone ERp29 : NMR structures of the domains and experimental model of the 51 kDa dimer

Funding Information: We thank Dr. William J. Griffiths for mass spectrometric analysis; Dr. Andrei Kaikkonen for the calculation of paramagnetic relaxation enhancements; and the Swedish NMR Center for access to the 800 MHz NMR spectrometer. This work was supported by the Swedish Natural Science Research Council, Swedish Medical Research Council, Swedish Society for Medical Research, and by an FRN grant for the 500 MHz NMR spectrometer. M.B. received a scholarship from the Swedish Institute and the Royal Swedish Academy. Copyright: Copyright 2012 Elsevier B.V., All rights reserved.Background: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. Results: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. Conclusions: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.publishersversionPeer reviewe