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.

α-Synuclein is a major component of Lewy bodies in Parkinson's disease. Although no signal sequence is apparent, α-synuclein expressed in Escherichia coli is mostly located in the periplasm. The possibilities that α-synuclein translocated into the periplasm across the inner membrane by the SecA or the Tat targeting route identified in bacteria and that α-synuclein was released through MscL were excluded. The signal recognition particle-dependent pathway is involved in the translocation of α-synuclein. The C-terminal 99-to-140 portion of the α-synuclein molecule plays a signal-like role for its translocation into the periplasm, cooperating with the central 61-to-95 section. The N-terminal 1-to-60 region is not required for this translocation.

No abstract

CheA-short interacts with CheZ to localize CheZ to cell poles. The fifth helical region (residues 112 to 133) from the phosphotransfer domain of CheA interacts with CheZ and becomes ordered and helical, although it lacks a stable fold in the CheA fragment comprising residues 98 to 150 alone. One CheA molecule binds to one CheZ dimer.During bacterial chemotaxis, transmembrane receptors regulate the activity of the chemotaxis-specific histidine autokinase CheA with the aid of a coupling protein, CheW. CheA acts to phosphorylate the response regulator CheY and the response regulator domain of the methylesterase CheB. Phosphorylated CheY (CheY-P) binds to the "switch complex" in the flagellar motor to regulate the sense of rotation of the motor. CheZ acts as a CheY phosphate phosphatase.Maddock and Shapiro (4) showed that the chemotaxis receptors tend to be clustered and often located at polar ends of bacterial cells. This localization of receptors is in large part dependent on the presence of CheA and CheW, and the clusters that form in wild-type cells contain receptors, CheA, CheW, CheY, and CheZ (8). These clusters are essential for proper communication among receptors and other members of the signal transduction complex.In Escherichia coli and many related bacteria, a naturally occurring short form of CheA (CheA S ) (7) interacts with CheZ, enhances the rate of dephosphorylation of CheY-P (5, 10), and is responsible for the localization of CheZ to the polar assemblies of receptors, CheA, and CheW (1). Having the kinase and the phosphatase colocalized generates more uniform CheY levels within the bacterial cell (9).In order to understand the structural basis of the CheA SCheZ interaction, we examined a CheA fragment containing residues 98 to 150 (CheA ). This fragment begins at the alternative site of translation initiation for CheA S and extends into the linker region joining the histidine phosphotransfer domain to the CheY-binding domain. This fragment includes residues that correspond to the C terminus of the fourth helix and the complete fifth helix of the intact histidine phosphotransfer domain, also known as the P1 domain. H dimension, suggesting a high degree of backbone mobility (2). Complete backbone assignments for the nonproline residues in CheA 98-150 were made using standard HNCACB and CBCA(CO)NH methods (6). We have assigned Glu100 through His154 (a residue of the His 6 tag). Although the nuclear magnetic resonance (NMR) spectra and 15 N relaxation properties (3) suggest that this fragment has no stable structure under these conditions, the central region (Asp112 to Glu133) exhibits positive ( 1 H-) 15 N nuclear Overhauser effects and large, positive (up to 2.6 ppm) C␣ secondary shifts (11), consistent with a partially rigid, helical structure.To establish which residues of CheA 98-150 are involved in CheZ binding, we titrated 15 N-labeled CheA 98-150 with unlabeled wild-type CheZ. The spectra were collected using a mixture of 50 mM sodium phosphate buffer, pH 6.8, 1 mM EDTA, and 2 mM dithiothreit...