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J. Biol. Chem., Vol. 280, Issue 14, 13529-13537, April 8, 2005

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Biophysical Characterization of ERp29

EVIDENCE FOR A KEY STRUCTURAL ROLE OF CYSTEINE 125^*

Veronique M. Hermann, John F. Cutfield, and Michael J. Hubbard¶||

From the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand and the ^¶Department of Paediatrics and School of Dental Science, The University of Melbourne, Melbourne, VIC 3052, Australia

ERp29 is a major resident of the endoplasmic reticulum (ER) that seemingly plays an important role in most animal cells. Although a protein-folding association is widely supported, ERp29's specific molecular function remains unknown. A chaperone activity was postulated from evidence that ERp29 forms multimers like the classical ER chaperones, but conflicting results have emerged from our recent studies. Here a biophysical approach was used to clarify this issue and also reveal a key structural role for ERp29's characteristic cysteine, Cys-125. Applying hydrodynamic parameters derived from sedimentation and dynamic light-scattering analyses, a model of ERp29's quaternary structure was assembled from existing tertiary substructures. Comparison with Windbeutel, an ERp29-like protein from fruit fly with specialized chaperone activity, revealed similar tri-lobar gross structures but some finer differences consistent with functional divergence. Solubility and hydrophobic probe assays revealed moderate surface hydrophobicity, which was reduced in mutant ERp29 in which serine replaced Cys-125. This mutant was also relatively labile to proteolytic degradation, providing two reasons for the strict conservation of Cys-125. No multimerization was observed with untagged ERp29, which existed as tight homodimers (K_d < 50 nM), whereas His-tagged ERp29 artifactually formed 670-kDa oligomers. These findings distinguish ERp29 biophysically from its peers in the ER including Windbeutel, endorsing our postulate that ERp29 adds a distinct type of folding activity to the ER machinery. By invoking novel functional associations for Cys-125 and the adjoining linker, new clues about how ERp29 might work have also arisen.

Received for publication, September 22, 2004 , and in revised form, November 5, 2004.

^* This work was supported in part by New Zealand Lottery Health Research and the Health Research Council of New Zealand (HRC(NZ)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A University of Otago Postgraduate Scholar.

^|| A Senior Research Fellow of the HRC(NZ). Recently supported by the Melbourne Research Unit for Facial Disorders and The University of Melbourne. To whom correspondence should be addressed: Dept. of Paediatrics, Royal Children's Hospital, Flemington Road, Melbourne, VIC 3052, Australia. Tel.: 61-3-9341-0449; Fax: 61-3-9341-0339; E-mail: mike.hubbard{at}unimelb.edu.au.

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