No abstract

Thioredoxin, DsbA, the N-terminal active-site domain a and the non-active-site domain b of protein-disulfide isomerase are all monomeric with a thioredoxin fold, and each exhibits low or no isomerase and chaperone activity. We have linked the N terminus of the above four monomers, individually, to the C terminus of the N-terminal domain of DsbC via the flexible linker helix of the latter to produce four domain hybrids, DsbCnTrx, DsbCn-DsbA, DsbCn-PDIa, and DsbCn-PDIb. These four hybrid proteins form homodimers, and except for DsbCn-PDIb they exhibit new or greatly elevated isomerase as well as chaperone activity. Three-dimensional structure prediction indicates that all the four domain hybrids adopt DsbC-like V-shaped structure with a broad uncharged cleft between the two arms for binding of non-native protein folding intermediates. The results provide strong evidence that dimerization creates chaperone and isomerase activity for monomeric thiol-protein oxidases or reductases, and suggesting a pathway for proteins to acquire new functions and/or higher biological efficiency during evolution.Many proteins, such as secretory proteins (antibodies, some peptide hormones) and membrane proteins (receptors, channel proteins), contain disulfide bonds, which play an essential role in stabilizing the tertiary and quaternary structures of these molecules. The formation of native disulfide bonds (including disulfide isomerization) is a key step in protein folding and is usually catalyzed by thiol-protein oxidoreductases, protein-disulfide isomerase (PDI) 1 in eukaryotes, and Dsb proteins in prokaryotes. So far at least six members of the Dsb family, DsbA, DsbB, DsbC, DsbD, DsbE, and DsbG, have been identified. In recent years PDI (1-4), DsbC (5), and DsbG (6) have been characterized to exhibit both disulfide isomerase and chaperone activity. The thiol-protein oxidoreductases contain thioredoxin (Trx) fold with one or more motif(s) of -CXYC-as active site(s). PDI is a homodimeric molecule mainly located within the endoplasmic reticulum, and each subunit is composed of four successive Trx-fold domains (a-b-bЈ-aЈ-) and a C-terminal tail (7). It is known that a and aЈ are homologous, and each has a -CGHC-motif as an active site. However, b and bЈ, without such an active site motif, are homologous with each other but not with the a domain. DsbC, located in the periplasm, is a prokaryotic counterpart of PDI and has been shown by crystal structure analysis (8) to be a V-shaped homodimer with each subunit forming an arm of the V. The N-terminal domain (1-61) of the subunit is linked via an ␣-helix-hinged linker (62-77) to the C-terminal Trx-domain (DsbCc, 78 -216) with a -CGYC-motif as an active site. The N-terminal domain from each monomer forms the dimer interface at the base of the V through ␤-sheet hydrogen bonds. The broad uncharged cleft with a large hydrophobic surface within the V has been suggested to be the site for peptide binding and therefore it plays a role in both the chaperone and the foldase activity of DsbC (8). B...

Escherichia coli DnaJ, possessing both chaperone and thiol-disulfide oxidoreductase activities, is a homodimeric Hsp40 protein. Each subunit contains four copies of a sequence of -CXXCXGXG-, which coordinate with two Zn(II) ions to form an unusual topology of two C4-type zinc fingers, C144DVC147Zn(II)C197NKC200 (Zn1) and C161PTC164Zn(II)C183PHC186 (Zn2). Studies on five DnaJ mutants with Cys in Zn2 replaced by His or Ser (C183H, C186H, C161H/C183H, C164H/183H, and C161S/C164S) reveal that substitutions of one or two Cys residues by His or Ser have little effect on the general conformation and association property of the molecule. Replacement of two Cys residues by His does not interfere with the zinc coordination. However, replacement of two Cys by Ser results in a significant decrease in the proportion of coordinated Zn(II), although the unique zinc finger topology is retained. The mutants of C183H, C186H, and C161S/C164S display full disulfide reductase activity of wild-type DnaJ, while C161H/C183H and C164H/183H exhibit severe defect in the activity. All of the mutations do not substantially affect the chaperone activity. The results indicate that the motif of -CXXC- is critical to form an active site and indispensable to the thiol-disulfide oxidoreductase activity of DnaJ. Each -CXXC- motif in Zn2 but not in Zn1 functions as an active site.