Substrate polypeptide presents a load on the apical domains of the chaperonin GroEL.

A conundrum has arisen in the study of the structural states of the GroEL-GroES chaperonin machine: When either ATP or ADP is added along with GroES to GroEL, the same asymmetric complex, with one ring in a GroES-domed state, is observed by either x-ray crystallographic study or cryoelectron microscopy. Yet only ATP/GroES can trigger productive folding inside the GroES-encapsulated cis cavity by ejecting bound polypeptide from hydrophobic apical binding sites during attendant rigid body elevation and twisting of these domains. Here, we show that this difference occurs because polypeptide substrate in fact presents a load on the apical domains, and, although ATP can counter this load effectively, ADP cannot. We monitored apical domain movement in real time by fluorescence resonance energy transfer (FRET) between a fixed equatorial fluorophore and one attached to the mobile apical domain. In the absence of bound polypeptide, addition of either ATP/GroES or ADP/GroES to GroEL produced the same rapid rate and extent of decrease of FRET (t(1/2) < 1 sec), reflecting similarly rapid apical movement to the same end-state and explaining the results of the structural studies, which were all carried out in the absence of substrate polypeptide. But in the presence of bound malate dehydrogenase or rhodanese, whereas similar rapid and extensive FRET changes were observed with ATP/GroES, the rate of FRET change with ADP/GroES was slowed by >100-fold and the extent of change was reduced, indicating that the apical domains opened in a slow and partial fashion. These results indicate that the free energy of gamma-phosphate binding, measured earlier as 43 kcal per mol (1 cal = 4.184 J) of rings, is required for driving the forceful excursion or "power stroke" of the apical domains needed to trigger release of the polypeptide load into the central cavity.