Topography of the Interaction of HPr(Ser) Kinase with HPr

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Abstract
The phosphocarrier protein, HPr, from Gram-positive organisms and mycoplasmas is a substrate for an ATP-dependent kinase that phosphorylates serine 46. In Gram-negative organisms, the corresponding HPr is not phosphorylated on serine 46 and the ATP-dependent kinase is absent. To determine the specificity requirements for phosphorylation of Mycoplasma capricolum HPr, a chimera in which residues 43−57 were replaced by the Escherichia coli sequence was constructed. The chimeric protein folded properly, but was not phosphorylated on either serine 46 or histidine 15. A dissection of the region required for phosphorylation specificity was carried out by further mutagenesis. The deficiency in phosphorylation at histidine 15 was localized primarily to the region including residues 51−57. Activity studies revealed that residues 48, 49, and 51−53 are important for recognition of M. capricolum HPr by its cognate HPr(Ser) kinase. The characteristics of this region suggest that the kinase−HPr interaction occurs mainly through a hydrophobic region. Molecular modeling comparisons of M. capricolum HPr and the chimeric construct provided a basis for interpreting the results of the activity assays.

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