The nucleotide-binding site of HisP, a membrane protein of the histidine permease. Identification of amino acid residues photoaffinity labeled by 8-azido-ATP.
The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP. These permeases are energized by ATP. HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport. In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP. HisP was derivatized with 8-azido-ATP (N3ATP). Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin. The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced. Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively. Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases. These consensus motifs are found in many purine nucleotide-binding proteins. The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed.
Mimura
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CS
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Admon
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Hurt
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Ames
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GF
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic; D001386:Azides; D001426:Bacterial Proteins; D001665:Binding Sites; D001693:Biological Transport, Active; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000032:analysis*; D006639:Histidine / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D014357:Trypsin
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D026923:Amino Acid Transport Systems, Basic; D001386:Azides; D001426:Bacterial Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009711:Nucleotides; D010446:Peptide Fragments; D006639:Histidine; C017040:8-azidoadenosine 5'-triphosphate; C043828:histidine permease, Bacteria; D000255:Adenosine Triphosphate; D014357:Trypsin