GroEL/GroES cycling: ATP binds to an open ring before substrate protein favoring protein binding and production of the native state

Abstract

The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide. In order-ofaddition experiments, approximately twice as much Rubisco activity was recovered when nonnative substrate protein was added after ATP compared with it being added before ATP, associated with twice as much Rubisco protein recovered with the chaperonin. Furthermore, the rate of Rubisco binding to an ATP-exposed ring was twice that observed in the absence of nucleotide. Finally, when both ATP and Rubisco were added simultaneously to a GroEL ring, simulating the physiological situation, the rate of Rubisco binding corresponded to that observed when ATP had been added first. We conclude that the physiological order, ATP binding before polypeptide, enables more efficient capture of nonnative substrate proteins, and thus allows greater recovery of the native state for any given round of the chaperonin cycle.chaperonin ͉ polypeptide binding ͉ protein folding T he GroEL/GroES chaperonin system provides assistance to folding of a large number of proteins to the native state via 2 principal actions, one involving binding of nonnative protein in an open ring of GroEL through multivalent hydrophobic contacts formed between the nonnative protein and surrounding GroEL apical domains and the other involving folding occurring in the encapsulated hydrophilic chamber formed when ATP and the GroES ''lid'' are bound to the same ring as polypeptide (1-3). A number of studies have made it clear that the polypeptide binding step can rescue misfolded substrate proteins from kinetically trapped states that occur during folding (e.g., 4, 5), despite the lack of stable secondary structure in such conformations (6, 7). Such rescue has been associated with topological ''stretching'' of the substrate protein, as observed in a number of FRET studies (5,8,9). Substrate protein is released during large ATP/GroES-directed rigid body movements of the GroEL apical domains into the GroES-domed hydrophilic chamber, in which it proceeds to fold (10-12). As shown in a number of studies, the so-called ''cis'' chamber facilitates folding by preventing multimolecular aggregation that could reduce both the rate of recovery of the native state (in those cases in which aggregation is reversible) and the extent of recovery (in those cases in which ag...

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

“…For example, recent work has suggested that non-native substrate protein can bind to the trans ring of an ATP bullet complex, an observation that conflicts with a central assumption of the standard reaction model (17). Other recent work has suggested that ADP release plays a central role in the reaction cycle (18,19), whereas still other studies have suggested that ATP binds to an open GroEL ring before non-native substrate protein (20). None of these observations are easily reconciled with the standard model of the GroEL reaction cycle.…”

Topographic Studies of the GroEL-GroES Chaperonin Complex by Chemical Cross-linking Using Diformyl Ethynylbenzene

“…This complex facilitates protein folding by sequestering polypeptide chains in a compartment formed by a nucleotide-bound, sevenmembered homooligomeric GroEL ring and a seven-membered homooligomeric GroES ring. An additional GroEL ring, the trans ring, sits adjacent to and facing the cis (GroESbound) ring but bears a collapsed conformation, which is unable to bind GroES until ATP hydrolysis and the completion of the catalytic cycle (33)(34)(35).…”

Dynamics, flexibility, and allostery in molecular chaperonins

“…It was long suggested that substrate binding precedes ATP binding and that, with associated apical domain movements, ATP binding has a role in protein unfolding; movements of the apical domains to which the protein substrate is attached force protein unfolding [47]. New data nonetheless suggest that ATP binding is more rapid than substrate association, indicating that ATP might already be bound when the substrate protein interacts with GroEL [48]. ATP addition does not appear to enhance substrate protein stretching, which would occur in a forced unfolding mechanism [48].…”

“…New data nonetheless suggest that ATP binding is more rapid than substrate association, indicating that ATP might already be bound when the substrate protein interacts with GroEL [48]. ATP addition does not appear to enhance substrate protein stretching, which would occur in a forced unfolding mechanism [48]. In contrast, the variety of The traditional model termed the ''bullet cycle'' assumes that only one folding chamber is active at a given time.…”