GroEL locked in a closed conformation by an interdomain cross-link can bind ATP and polypeptide but cannot process further reaction steps

It has been believed that when GroEL binds to GroES its apical domain moves upward and outward, To inhibit this ''opening'' movement, its equatorial and apical domains were cross-linked through a disulfide bond between mutationally introduced cysteine residues at the positions of Asp-83 and Lys-327. To avoid possible undesired cross-linking, we at first prepared a mutant GroEL (GroEL(NC); Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser) in which all cysteine residues in wild-type GroEL were replaced by serine residues, GroEL(NC) was fully functional as a chaperonin, We then introduced the above two point mutations into GroEL(NC), to generate a mutant (GroEL(AEX); Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser and Asp-83 --> Cys, Lys 327 --> Cys). Oxidized GroEL(AEX), which is locked in a ''closed'' conformation by an interdomain disulfide bond, can bind 6-7 mol of ATP, which remain bound without hydrolysis, This ATP-bound, oxidized GroEL(AEX) can bind the stably nonnative substrate protein isopropylmalate dehydrogenase, whereas the nucleotide-free oxidized GroEL(AEX) binds it with a weaker affinity, However, oxidized GroEL(AEX) fails to process further reaction steps such as ATP hydrolysis, binding of GroES, dissociation of substrate protein from GroEL, and facilitating protein folding, When disulfide bonds in oxidized GroEL(AEX) are reduced, GroEL(AEX) exerts the ability to process all the reactions just as GroEL(NC) and wild-type GroEL. Indications from these results are: hydrolysis of ATP may require opening movement of the apical domain; GroES binds to an open form of GroEL; and substrate polypeptide is released from GroEL coupled with either ATP hydrolysis or opening movement of the apical domain.