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

Abstract

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 ATP͞ 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 ATP͞GroES or ADP͞GroES 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 ATP͞GroES, the rate of FRET change with ADP͞GroES 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 ␥-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.

Single-molecule spectroscopy of protein folding in a chaperonin cage

“…5C). The rate constant for the initial decrease of 7 AE 2 s −1 is close to the value reported for GroESbinding (19 s −1 ) under these conditions (48), and the slower increase can be described with the reported rate of apical domain movement of SR1 under substrate load of 0.68 s −1 (48). These changes in the average transfer efficiency probably reflect very small conformational rearrangements of the substrate or the fluorophores during encapsulation, which are unlikely to be able to cause a selective deceleration of folding of the C-terminal domain inside the chaperonin cavity on the time scale of minutes to hours.…”

Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state

“…Initial docking of GroES is then followed by large rigid body apical domain movements, 60°elevation and 120°clockwise twist, which produce the stable domed GroESencapsulated end state that has been observed by both cryo-EM and X-ray studies (4,5,9). Notably, this conformational progression and end state, produced in Ͻ1 s in ATP, can be almost as rapidly driven in the nonphysiologic nucleotide, ADP, as long as substrate polypeptide, which acts as a retarding ''load'' on apical domain movement, is not present (18). Thus, ADP appears in this context to be an informative nucleotide concerning GroES binding.…”

“…We surmised that whereas GroES could bind to these complexes, the substrate polypeptide had failed to be ejected off of the cavity wall into the folding chamber. Such behavior resembles that observed when, for example, GroEL-rhodanese binary complexes are incubated with ADP and GroES, forming a substrate-GroEL-GroES ternary complex in which rhodanese remains bound to the GroEL cavity wall (7,18). To assess here whether polypeptide remained associated with the cavity wall of the mixed-ring complex, we asked whether it could transfer to an added GroEL ''trap'' molecule upon ATP-GroES binding (Fig.…”

“…In particular, the apical domains of GroEL are observed to move more slowly in the presence of bound substrate protein during the formation of ADP-GroES complexes, failing to release the substrate, reflecting that ADP-driven movements may not be as forceful (18). In addition, ATP binds cooperatively to GroEL whereas ADP does not (19).…”

“…ATP is specifically required for the activation of folding (16,17), with ADP unable to support release of polypeptide into the folding chamber despite the ability to produce the movements that enable stable binding of GroES (7,9,11,18). In particular, the apical domains of GroEL are observed to move more slowly in the presence of bound substrate protein during the formation of ADP-GroES complexes, failing to release the substrate, reflecting that ADP-driven movements may not be as forceful (18).…”