Topography of the interaction of HPr(Ser) kinase with HPr.
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.
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Zhu
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Herzberg
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Peterkofsky
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009174:Mycoplasma / Q000201:enzymology / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D012694:Serine; D006639:Histidine; D001216:Asparagine; D011494:Protein Kinases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases