Identification of nucleotide-binding regions in the chaperonin proteins GroEL and GroES.
The chaperonin GroEL, a tetradecameric cylinder consisting of subunits of M(r) approximately 60,000 (60K), and its cofactor GroES, a heptameric ring of 10K subunits, mediate protein folding in the cytosol of Escherichia coli. In the presence of nucleotide, GroES forms a 1:1 complex with GroEL which binds unfolded protein in its central cavity and releases it to allow folding upon ATP hydrolysis. Using labelling with azido-ATP, we have identified a protease-stable nucleotide-binding domain of M(r) 40K in the GroEL subunits (residues 153-531). Azido-ATP is crosslinked to the highly conserved Tyr 477, indicating that this residue is close to the purine ring of the bound nucleotide. Surprisingly, GroES also binds ATP cooperatively and with an affinity comparable to that of GroEL. Azido-nucleotide labelling of GroES subunits occurs at the conserved Tyr 71 in a protease-stable 6.5K domain (starting at residue 33). Proteinase K cleavage at residue 32 is prevented when GroES is bound to GroEL. ATP binding to GroES may be important in charging the seven subunits of the interacting GroEL ring with ATP to facilitate cooperative ATP binding and hydrolysis for substrate protein release.
Martin
J
J
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021.
Geromanos
S
S
Tempst
P
P
Hartl
F U
FU
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D018835:Chaperonin 10; D018834:Chaperonin 60; D004926:Escherichia coli / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D009711:Nucleotides; D014443:Tyrosine; D000255:Adenosine Triphosphate