Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL.
A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.
Nojima
Tatsuya
T
Chemical Resources Laboratory R1-7, Tokyo Institute of Technology, Yokohama, Japan.
Murayama
Shigeto
S
Yoshida
Masasuke
M
Motojima
Fumihiro
F
D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D018835:Chaperonin 10 / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013379:Substrate Specificity; D013997:Time Factors
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D049452:Green Fluorescent Proteins; D008291:Malate Dehydrogenase