Identification of amino acid residues at nucleotide-binding sites of chaperonin GroEL/GroES and cpn10 by photoaffinity labeling with 2-azido-adenosine 5'-triphosphate.
Although the chaperonin GroEL/GroES complex binds and hydrolyzes ATP, its structure is unlike other known ATPases. In order to better characterize its nucleotide binding sites, we have photolabeled the complex with the affinity analog 2-azido-ATP. Three residues of GroEL, Pro137, Cys138 and Thr468, are labeled by the probe. The location of these residues in the GroEL crystal structure [Braig, K., Otwinowski, Z., Hedge, R., Boisvert, D., Joachimiak, A., Horwich, A. & Sigler, P. (1994) Nature 371, 578-586: Boisvert, D. C., Wang, J., Otwinowski, Z., Horwich, A. L. & Sigler, P. B. (1996) Nat. Struct. Biol. 3, 170-177] suggests that 2-azido-ATP binds to an alternative conformer of GroEL in the presence of GroES. The labeled site appears to be located at the GroEL/GroEL subunit interface since modification of Pro137 and Cys138 is most readily explained by attack of a probe molecule bound to the adjacent GroEL subunit. Labeling of the co-chaperonin, GroES, is clearly demonstrated on gels and the covalent tethering of nucleotide allows detection of a GroES dimer in the presence of SDS. However, no stable peptide derivative of GroES could be purified for sequencing. In contrast, the GroES homolog, yeast cpn10, does give a stable derivative. The modified amino acid is identified as the conserved Pro13, which corresponds to Pro5 in Escherichia coli GroES.
Bramhall
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EA
Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center at Syracuse 13210, USA.
Cross
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RL
Rospert
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Steede
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NK
Landry
S J
SJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D001386:Azides; D001665:Binding Sites; D018835:Chaperonin 10 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D018835:Chaperonin 10; D018834:Chaperonin 60; D009711:Nucleotides; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate