Rational mutagenesis of a 40 kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function

Hennessy, Fritha; Boshoff, Aileen; Blatch, Gregory L.

doi:10.1016/j.biocel.2004.06.009

Cited by 35 publications

(31 citation statements)

References 49 publications

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“…Apart from the HPD motif, the other amino acids on the J-domain of Hsp40 proteins that are involved in the binding to a partner Hsp70 are less precisely defined. However, as a result of our work (Hennessy, Cheetham, Dirr, & Blatch, 2000; Hennessy, Boshoff, & Blatch, 2005a) and that of other researchers (Garimella et al, 2006; Genevaux, Schwager, Georgopoulos, & Kelley, 2002; Genevaux et al, 2003; Lu & Cyr, 1998; Suh et al, 1999), other residues and regions outside the HPD motif, especially helices II, III and IV, are gradually being implicated in the general binding and specificity of interaction of Hsp40 proteins with Hsp70 proteins.…”

Section: Introductionsupporting

confidence: 61%

“…Leu10 projects toward the helix II–helix III inter-helical space, potentially interacting with residues of helices II and III. Therefore, Tyr7 and Leu10 may be critical in ensuring the stability of the helix-loop structure of helix II–helix III for presentation to Hsp70 (Hennessy et al, 2005a). Arg26 has been shown to be critical for J-domain function in E. coli DnaJ and Agrobacterium tumefaciens DnaJ (Agt DnaJ), and has been proposed to be part of a network of residues on helix II (and possibly helix III) that form an Hsp70-binding site on the J-domain (Genevaux et al, 2002; Hennessy et al, 2005a,b).…”

Section: Introductionmentioning

confidence: 99%

“…In particular, the F47A substitution in the J-domain of E. coli DnaJ resulted in loss of in vivo function (Genevaux et al, 2002), suggesting this was an essential amino acid for J-domain structure and function, potentially by forming interactions with His33 of the HPD motif. Interestingly, an F47L substitution in Agt DnaJ had no detectable effect on its in vivo function, suggesting that the Leu residue could sufficiently contribute to the contacts originally made by the Phe residue (Hennessy et al, 2005a). The highly conserved Leu57 of helix III projects into the J-domain interior and is likely to be a key residue in holding helices II and III together.…”

Section: Introductionmentioning

confidence: 99%

“…The highly conserved Leu57 of helix III projects into the J-domain interior and is likely to be a key residue in holding helices II and III together. Furthermore, experimental evidence has suggested that Leu57 was essential for J-domain function (Hennessy et al, 2005a). While residues of helices II and III and the loop region linking the two helices are crucial to J-domain function (Pellecchia et al, 1996), it has been proposed that the residues of helix IV are not essential to the co-chaperone function of DnaJ (Genevaux et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Nicoll

Botha

McNamara

et al. 2007

The International Journal of Biochemistry & Cell Biology

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Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I–IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.

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Section: Introductionsupporting

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Nicoll

Botha

McNamara

et al. 2007

The International Journal of Biochemistry & Cell Biology

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“…The HPD motif is required for the stimulation of the ATPase activity of the partner Hsp70 chaperone. Mutations of this conserved motif render the Hsp40 incapable of stimulating the ATPase activity of Hsp70 above basal levels and therefore impair co-chaperone activity [31]. While the J domain is an essential part of the Hsp40 proteins, the other domains have important functions as well.…”

Section: Structure and Classification Of Hsp40mentioning

confidence: 99%

Hsp40 Co-chaperones as Drug Targets: Towards the Development of Specific Inhibitors

Pesce

Blatch

Edkins

2015

Topics in Medicinal Chemistry

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The heat shock protein 40 (Hsp40/DNAJ) family of co-chaperones modulates the activity of the major molecular chaperone heat shock protein 70 (Hsp70) protein group. Hsp40 stimulates the basal ATPase activity of Hsp70 and hence regulates the affinity of Hsp70 for substrate proteins. The number of Hsp40 genes in most organisms is substantially greater than the number of Hsp70 genes. Therefore, different Hsp40 family members may regulate different activities of the same Hsp70. This fact, along with increasing knowledge of the function of Hsp40 in diseases, has led to certain Hsp40 isoforms being considered promising drug targets. Here we review the role of Hsp40 in human disease and recent developments towards the creation of Hsp40-specific inhibitors.

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Sequence and domain conservation of the coelacanth Hsp40 and Hsp90 chaperones suggests conservation of function

2013

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Molecular chaperones and their associated co-chaperones play an important role in preserving and regulating the active conformational state of cellular proteins. The chaperone complement of the Indonesian Coelacanth, Latimeria menadoensis, was elucidated using transcriptomic sequences. Heat shock protein 90 (Hsp90) and heat shock protein 40 (Hsp40) chaperones, and associated co-chaperones were focused on, and homologous human sequences were used to search the sequence databases. Coelacanth homologs of the cytosolic, mitochondrial and endoplasmic reticulum (ER) homologs of human Hsp90 were identified, as well as all of the major co-chaperones of the cytosolic isoform. Most of the human Hsp40s were found to have coelacanth homologs, and the data suggested that all of the chaperone machinery for protein folding at the ribosome, protein translocation to cellular compartments such as the ER and protein degradation were conserved. Some interesting similarities and differences were identified when interrogating human, mouse, and zebrafish homologs. For example, DnaJB13 is predicted to be a non-functional Hsp40 in humans, mouse, and zebrafish due to a corrupted histidine-proline-aspartic acid (HPD) motif, while the coelacanth homolog has an intact HPD. These and other comparisons enabled important functional and evolutionary questions to be posed for future experimental studies.

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Rational mutagenesis of a 40 kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function

Cited by 35 publications

References 49 publications

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Hsp40 Co-chaperones as Drug Targets: Towards the Development of Specific Inhibitors

Sequence and domain conservation of the coelacanth Hsp40 and Hsp90 chaperones suggests conservation of function

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