The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties.
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 10kDa C-terminal alpha-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal alpha-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa. A.Boshoff@ru.ac.za
Aileen
A
Boshoff
Linda L
LL
Stephens
Gregory L
GL
Blatch
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D005816:Genetic Complementation Test; D006868:Hydrolysis; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases