The International Journal of Biochemistry & Cell Biology 40 (2008) 804–812 Available online at www.sciencedirect.com The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties Aileen Boshoff ∗,1 , Linda L. Stephens 1 , Gregory L. Blatch Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa Received 24 August 2007; received in revised form 15 October 2007; accepted 15 October 2007 Available online 23 October 2007 Abstract 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 -helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal -helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation. © 2007 Elsevier Ltd. All rights reserved. Keywords: Agrobacterium tumefaciens; Escherichia coli; DnaK; ATPase; Thermal aggregation; Oligomerisation 1. Introduction In response to stress, cells increase the synthesis of heat shock proteins in order to cope with increased Abbreviations: Agt, Agrobacterium tumefaciens; A. tumefaciens, Agrobacterium tumefaciens; Heat shock protein 70, Hsp70; ATP, adenosine triphosphate; His, histidine; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography. ∗ Corresponding author. Fax: +27 46 622 3984. E-mail address: A.Boshoff@ru.ac.za (A. Boshoff). 1 These authors contributed equally to the research. amounts of denatured proteins. The heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) of molecular chaperones plays a key role in the net- work of cellular chaperones. Hsp70 facilitates folding of nascent polypeptides (Eggers, Welch, & Hansen, 1997), assists in protein translocation (Hamman, Hendershot, & Johnson, 1998) and assembly of protein complexes (Zylicz, Ang, Liberek, & Georgopoulos, 1989), pre- vents protein aggregation by binding to hydrophobic segments of unfolded proteins, and is involved in tar- geting proteins for degradation (Brodsky & McCracken, 1999). The Hsp70 protein functions both as a holdase (binding and holding onto unfolded polypeptides by 1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.10.017