Contribution of the HEDJ/ERdj3 cysteine-rich domain to substrate interactions.
Cytoplasmic type I DnaJ/Hsp40 chaperones contain a Cys-rich domain consisting of four CXXCXG motifs that are in a reduced state and coordinate zinc, stabilizing the intervening sequence in a loop structure. However, the Cys-rich region of the endoplasmic reticulum localized HEDJ (ERdj3/ERj3p), is considerably different in sequence and arrangement. Unlike the typical type I molecule, the HEDJ CXC, and CXXC motifs were demonstrated in this study to be predominantly oxidized in intramolecular disulfide bonds. In the native state, HEDJ bound to immobilized, denatured thyroglobulin. Unlike its binding partner GRP78, redox conditions affected the interaction of HEDJ with substrate. Substitution of the Cys-rich domain cysteine residues with serine diminished or abolished HEDJ binding in the in vitro assay. These findings suggest that the Cys-rich region of HEDJ and its oxidation state are important in maintaining the substrate interaction domain in a binding-competent conformation.
Marcus
Nancy Y
NY
Department of Pediatrics and Molecular Microbiology, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110, USA. nmarcus@slu.edu
Marcus
Roland A
RA
Schmidt
Bela Z
BZ
Haslam
David B
DB
D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D008861:Microsomes / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
C494648:DNAJB11 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D050956:HSP40 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D003545:Cysteine