Dimerization by Domain Hybridization Bestows Chaperone and Isomerase Activities

Self Cite

DnaJ, an Escherichia coli Hsp40 protein composed of 376 amino acid residues, is a chaperone with thioldisulfide oxidoreductase activity. We present here for the first time a small angle x-ray scattering study of intact DnaJ and a truncated version, DnaJ (1-330), in solution. The molecular weight of DnaJ and DnaJ (1-330) determined by both small angle x-ray scattering and size-exclusion chromatography provide direct evidence that DnaJ is a homodimer and DnaJ (1-330) is a monomer. The restored models show that DnaJ is a distorted -shaped dimeric molecule with the C terminus of each subunit forming the central part of the , whereas DnaJ (1-330) exists as a monomer. This indicates that the deletion of the C-terminal 46 residues of DnaJ impairs the association sites, although it does not cause significant conformational changes. Biochemical studies reveal that DnaJ (1-330), while fully retaining its thiol-disulfide oxidoreductase activity, is structurally less stable, and its peptide binding capacity is severely impaired relative to that of the intact molecule. Together, our results reveal that the C-terminal (331-376) residues are directly involved in dimerization, and the dimeric structure of DnaJ is necessary for its chaperone activity but not required for the thiol-disulfide oxidoreductase activity.Hsp40 proteins collaborate specifically with Hsp70 proteins and some other factors to participate in a wide range of cellular processes essential to cell survival, such as assisting the folding, assembly, disassembly, and translocation of newly synthesized proteins, by suppressing aggregation or mediating degradation of misfolded proteins (1, 2). The Hsp40 family proteins are divided into three subtypes (3) (see Scheme 1). They all contain a J domain responsible for binding to the ATPase domain and stimulating the ATPase activity of Hsp70. Type I Hsp40 proteins, such as Escherichia coli DnaJ, yeast Yjd1, and human Hdj-2, have a Gly/Phe-rich region, which has been suggested to assist J domain interactions with Hsp70 (4). The Gly/Phe-rich region is followed by a conserved cysteine-rich region forming a zinc finger domain and a poorly conserved C-terminal domain. The peptide binding site(s) has been found to locate within these two domains (5-7). Type II Hsp40 proteins, such as yeast Sis1 and mammalian Hdj-1, also contain the J, a Gly/Phe-rich region, and C-terminal domains but without the zinc finger domain (8, 9). Both type I and type II Hsp40 proteins act as molecular chaperones to bind and deliver nonnative proteins to Hsp70 (7, 8, 10), but they are not functionally equivalent (11). Type III Hsp40 proteins, such as yeast YJL162c and virus T antigen, contain only the J domain, and they do not function as molecular chaperones (3).In addition to the DnaK-dependent co-chaperone activity described above, DnaJ also exerts autonomous DnaK-independent chaperone activity (9, 12, 13), which is characterized by its ability to bind with unfolded proteins and prevent them from irreversible aggregation (10, 11). Moreover, DnaJ has ...

Section: Discussionmentioning

confidence: 56%

The C-terminal (331–376) Sequence of Escherichia coli DnaJ Is Essential for Dimerization and Chaperone Activity

Shi

Hong

Wang

2005

Self Cite

“…Furthermore, inclusion of the Y55K mutation into monomeric Wind (D31N/ R41S) does not lead to significantly enhanced substrate binding in comparison to Y55K alone. A requirement for dimerization or for the participation of two or more, covalently linked thioredoxin domains in chaperone/redox activity has also been noted for bacterial DsbC and PDI (7,17,18).…”

Section: Fig 8 D-domain Mutants Affect Pipe Processingmentioning

confidence: 90%

Mapping of a Substrate Binding Site in the Protein Disulfide Isomerase-related Chaperone Wind Based on Protein Function and Crystal Structure

Barnewitz

Guo

Sevvana

et al. 2004

The protein disulfide isomerase (PDI)-related protein Wind is essential inPDI 1 -related proteins are residents of the lumen of the endoplasmic reticulum (ER) with various functions, including redox and chaperone activities, regulation of calcium homeostasis, and regulation of protein export from the ER for degradation. These functions are essential for maintaining a productive folding environment for many secretory proteins within the ER (1, 2) that may be critical for viability of the organism (3, 4). The chaperone function of these proteins, and probably to varying extents redox activity as well, relies on their ability to interact non-covalently with specific peptide sequences or epitopes in substrate proteins (2). However, until recently no concise data on peptide specificity, peptide binding sites, or the molecular basis for the observed substrate selectivity of these proteins were available. Data on such inherently weak interactions would require detailed knowledge of the three-dimensional structure of the protein concerned. Although independent NMR structures of the isolated a-and b-type thioredoxin fold domains (redox-active and redox-inactive domains, respectively) of PDI have been reported (5, 6), no complete structure of a eukaryotic PDI protein was available. Most PDI proteins are redox-active, and the involvement of relatively strong heteromeric disulfide bond formation and reduction in assays aimed at elucidating the nature of weak chaperone interactions poses further problems. Because the bЈ-domain has been identified as the major substrate binding site in PDI (7), much attention has been focused on these redox-inactive domains. However, the lack of suitable substrates of physiological relevance with a maturation process that can be readily monitored has complicated these studies. Thus much could be learned from the study of peptide binding and chaperone activity of a naturally occurring PDI family protein lacking redox properties but having a clearly defined substrate that can be studied both in vivo and in vitro.We recently described the first crystal structure of a complete PDI-related protein of the eukaryotic ER (3). This protein, Wind, is essential in Drosophila melanogaster for dorsal-ventral patterning within the developing embryo, and it is required for ER export of an essential Golgi transmembrane proteoglycan modifying enzyme, Pipe (a 2-O-sulfotransferase) (8). Apart from a b-domain, Wind also has a unique C-terminal domain found only in the PDI-D subclass of PDI-related proteins (1). The function of the D-domain is poorly understood, although in Dictyostelium discoideum PDI-D it was shown to play a role in ER retention (9). Recently, we have shown that the Pipe-processing activity requires both the b-and D-domains of Wind. Although the mammalian Wind orthologue ERp28/29 cannot replace Wind in Pipe processing, the D-domains of both proteins could be exchanged, indicating functional conservation between the proteins (3).Here, we show that Wind binds Pipe directly in vitro, and we map a ...

“…In addition, DsbH was unable to complement for DsbC in copper resistance assays (Table 2), showing the absence of isomerase functionality also in vivo. In addition, dimerization is known to be very important for disulfide isomerase activity (47,48), but DsbH is a monomer in solution. Thus, in accordance with its redox potential, DsbH appears to be a periplasmic reductase.…”

Section: Resultsmentioning

confidence: 99%

Insight into Disulfide Bond Catalysis in Chlamydia from the Structure and Function of DsbH, a Novel Oxidoreductase

Mac

Hacht

Hung

et al. 2008

The Chlamydia family of human pathogens uses outer envelope proteins that are highly cross-linked by disulfide bonds but nevertheless keeps an unusually high number of unpaired cysteines in its secreted proteins. To gain insight into chlamydial disulfide bond catalysis, the structure, function, and substrate interaction of a novel periplasmic oxidoreductase, termed DsbH, were determined. The structure of DsbH, its redox potential of ؊269 mV, and its functional properties are similar to thioredoxin and the C-terminal domain of DsbD, i.e. characteristic of a disulfide reductase. As compared with these proteins, the two central residues of the DsbH catalytic motif (CMWC) shield the catalytic disulfide bond and are selectively perturbed by a peptide ligand. This shows that these oxidoreductase family characteristic residues are not only important in determining the redox potential of the catalytic disulfide bond but also in influencing substrate interactions. For DsbH, three functional roles are conceivable; that is, reducing intermolecular disulfides between proteins and small molecules, keeping a specific subset of exported proteins reduced, or maintaining the periplasm of Chlamydia in a generally reducing state.Chlamydia are obligate intracellular eubacteria that are phylogenetically distant from other bacterial divisions (1). Virtually every human being will be infected with Chlamydia pneumoniae in their lifetime, and this organism accounts for ϳ10% of pneumonia cases and 5% of bronchitis cases in the United States (2, 3). Acute infections are usually mild in immunocompetent hosts, but severe pneumonias are observed in immunocompromised patients. Chronic C. pneumoniae infections have been associated with arterial disease (4), where C. pneumoniae act to continuously stimulate inflammation and thereby contribute to stroke and coronary heart disease. Chlamydia have an unique developmental cycle in which the extracellular infectious form, the elementary body, is metabolically inactive and highly resistant to lysis. This is thought to be due in large part to a complex of outer envelope proteins that are highly crosslinked by disulfide bonds (5). Disulfide cross-linked envelope proteins appear to substitute for peptidoglycan in the elementary body (6). This is in contrast to the intracellular, non-infectious but metabolically active reticulate body which employs peptidoglycan (7), like other Gram-negative bacteria. The reliance of the elementary body on disulfide bond-linked proteins suggests that interference with chlamydial disulfide bond catalysis offers a potential avenue for treating chlamydial infections in addition to antibiotics administration. Despite the extensive use of disulfide cross-linked envelope proteins, Chlamydia exhibit an unusually high number of unpaired cysteines in their secreted proteins compared with Escherichia coli. Is the machinery for periplasmic disulfide bond catalysis in Chlamydia different from E. coli?Functional gene assignment of the C. pneumoniae TW183 genome predicts 5 periplasmic...