Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics Philip Peake a;b; *, Nathalie Winter 1; a , Warwick Britton a; c a Centenary Institute for Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW 2042, Australia b Sutherland Centre of Immunology, Sutherland Hospital, Caringbah 2229, Australia c Department of Medicine, University of Sydney, NSW 2006, Australia Received 13 May 1998; revised 24 June 1998; accepted 1 July 1998 Abstract We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue. Recombinant M. leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively. Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates. Autophosphorylation was augmented by up to 25 mM Ca 2 . Using site-directed mutagenesis to construct two ThrCAla mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr 175 was the dominant threonine residue autophosphorylated in M. leprae Hsp70. Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin. These properties are compared with those of the DnaK protein of Escherichia coli. ß 1998 Elsevier Science B.V. All rights reserved. Keywords : DnaK; Heat shock; Hsp70; Autophosphorylation; (Mycobacterium leprae) 1. Introduction The heat shock response is ubiquitous in nature. The 70 kDa family of heat-shock proteins (Hsp70) are present in both pro- and eukaryotes, and display a high degree of structural and functional homology. Their roles include binding to unfolded or nascent polypeptides, and the renaturation and disaggrega- tion of misfolded polypeptides. Members of the Hsp70 family also participate in replication and pro- tein transport within the cell [1^4]. The highly con- served N-terminal region of Hsp70 from all species described so far possesses a weak adenosine triphos- phatase (ATPase) activity which is essential for many of these activities. The less well conserved C-terminus of the molecule contains the substrate binding region [4,5]. Mutations in the DnaK gene of Escherichia coli, which produces a homologue of Hsp70, have pleiotropic e¡ects on such processes as the autoregu- lation of the heat-shock response, DNA replication, synthesis of host RNA and cell division [6^9]. Under certain conditions autophosphorylation occurs, but the e¡ect this may have on the structure and function of the molecule is unclear. In mycobacteria, Hsp70 homologues have at- tracted attention as they are dominant antigens for both T- and B-cell responses in man and infected 0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII:S0167-4838(98)00156-3 * Corresponding author. Address b. Fax : +61-2-9540-7411. 1 Present address : Unit de Genetique Mycobacterienne, Institut Pasteur, Paris, France. Biochimica et Biophysica Acta 1387 (1998) 387^394