The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties

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Boshoff, Aileen, Stephens, L and Blatch, Gregory ORCID: 0000-0003-0778-8577 (2008) The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties. International Journal of Biochemistry and Cell Biology, 40 (4). pp. 804-812. ISSN 1357-2725


DnaK is a molecular chaperone that promotes cell survival during stress by preventing protein misfolding. The chaperone activity is regulated by nucleotide binding and hydrolysis events in the N-terminal ATPase domain, which in turn mediate substrate binding and release in the C-terminal substrate binding domain. In this study we determined that ATP hydrolysis was the rate limiting step in the ATPase cycle of Agrobacterium tumefaciens DnaK (Agt DnaK); however the data suggested that Agt DnaK had a significantly lower affinity for ATP than Escherichia coli DnaK. We show for the first time that Agt DnaK was very effective at preventing thermal aggregation of malate dehydrogenase (MDH) in a concentration dependent manner. This is in contrast to E. coli DnaK which was ineffective at preventing thermal aggregation of MDH. A mutant Agt DnaK-V431F, with a blocked hydrophobic pocket in the substrate binding domain, was unable to suppress the thermosensitivty of an E. coli dnaK103 deletion strain. However the mutation did not inhibit Agt DnaK-V431F from preventing the thermal aggregation of MDH. The oligomeric state of Agt DnaK was studied using size exclusion chromatography. We demonstrated that dilution of the Agt DnaK protein, the addition of ATP and the removal of the 10 kDa C-terminal α-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal α-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.

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Online ISSN: 1878-5875

Item type Article
DOI 10.1016/j.biocel.2007.10.017
Official URL
Subjects Historical > Faculty/School/Research Centre/Department > School of Biomedical and Health Sciences
Historical > FOR Classification > 0601 Biochemistry and Cell Biology
Historical > FOR Classification > 0604 Genetics
Keywords ResPubID22204. Agrobacterium tumefaciens, Escherichia coli, DnaK, ATPase, thermal aggregation, oligomerisation, chaperones
Citations in Scopus 8 - View on Scopus
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