Crystal structure of a GroEL-ADP complex in the relaxed allosteric state at 2.7 Å resolution

Abstract

The chaperonin proteins GroEL and GroES are cellular nanomachines driven by the hydrolysis of ATP that facilitate the folding of structurally diverse substrate proteins. In response to ligand binding, the subunits of a ring cycle in a concerted manner through a series of allosteric states (T, R, and R″), enabling work to be performed on the substrate protein. Removing two salt bridges that ordinarily break during the allosteric transitions of the WT permitted the structure of GroEL-ADP in the R state to be solved to 2.7 Å resolution. Whereas the equatorial domain displays almost perfect sevenfold symmetry, the apical domains, to which substrate proteins bind, and to a lesser extent, the intermediate domains display a remarkable asymmetry. Freed of intersubunit contacts, the apical domain of each subunit adopts a different conformation, suggesting a flexibility that permits interaction with diverse substrate proteins. This result contrasts with a previous cryo-EM study of a related allosteric ATP-bound state at lower resolution. After artificially imposing sevenfold symmetry it was concluded that a GroEL ring in the R-ATP state existed in six homogeneous but slightly different states. By imposing sevenfold symmetry on each of the subunits of the crystal structure of GroEL-ADP, we showed that the synthetic rings of (X-ray) GroEL-ADP and (cryo-EM) GroEL-ATP are structurally closely related. A deterministic model, the click stop mechanism, that implied temporal transitions between these states was proposed. Here, however, these conformational states are shown to exist as a structurally heterogeneous ensemble within a single ring.protein machine | allostery | chaperonin T o assist protein folding, the GroEL/GroES chaperonin machine cycles through a series of conformational states in response to ligand binding (1-3). Two states, taut (T) and relaxed (R), are populated in the absence of GroES. Another two states, R′ and R″, that exist before and after ATP hydrolysis, respectively, are populated on GroES binding to the R state (1-3) (Fig. 1A). Crystal structures of T [Protein Data Bank (PDB) ID code 1OEL] (4) (PDB ID code 1XCK) (5), R′ (PDB ID code 1SVT) (6), and R″ (PDB ID code 1AON) (7) states are available.As described by the theory of nested cooperativity, the conformational transitions within each heptameric ring are positively cooperative, whereas the transitions between the rings are negatively cooperative (8, 9). Multiple salt bridges, some within a subunit and others between subunits, stabilize these various conformational states. In such a dynamic system, these salt bridges must continually be broken and reformed at different points during the chaperonin cycle. Two interdomain salt bridges, an intrasubunit one (D83-K327) and an intersubunit one (R197-E386), stabilize the T state and are ordinarily broken during the T to R transition that follows the binding of ATP to the WT (Fig. 1B). Individually, the R197A mutation destabilizes the T state (10, 11), whereas replacing the D83-K327 salt bridge with a disulfid...

Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

“…This distance variability in D83-K327 is similar to that among the matched crystal structures (16). Interestingly, these residues are located in the close proximity of the entry path of ATP to its binding pocket (24). Thus, D83-K327 could be a chemical switch or sensor linking the apical domain flexibility and the ATP-binding pocket.…”

“…High apical-domain ADP distributions have been found in multiple crystal structures (Fig. S3B) (15,24) and molecular dynamics simulations of apo-GroEL (22). Thus, localized flexibility of the GroEL apical domain is an inherent Significance Using cryo-EM and expanding on focused classification allowed for the bacterial chaperone, GroEL, to be structurally resolved at atomic detail one particle at a time.…”

GroEL2 of Mycobacterium tuberculosis Reveals the Importance of Structural Pliability in Chaperonin Function

“…Binding of ATP triggers breaking of several intra-and intersubunit salt bridges between the equatorial and intermediate domains, resulting in a 25°movement of intermediate domain through hinge 1 (18)(19)(20). This imparts strain on the salt bridges between the apical and intermediate domains, which eventually break and release the apical domain for large rotations across hinge 2 (21,42). The hinge regions are thus critical in the GroEL mechanism, and variations in hinge sequences can potentially affect one or more features of the GroEL mechanism.…”

“…The three-domain architecture of the GroEL protomer principally adopts two conformations: polypeptide-accepting T state and nucleotide-bound RЉ state. The T and RЉ conformations are observed in trans and cis rings in the GroES-GroEL complex (42). Transition between the two conformations essentially involves communication between the equatorial domain (E) and apical domain (A) in response to the presence of nucleotide.…”

Asymmetric perturbations of signalling oligomers

“…SAXS could distinguish between the biological relevance of symmetric and asymmetric configurations of the GroEL-GroES complex (Inobe et al, 2008). A recent comparative XRC and cryo-EM study has revealed that functional transition of GroEL into a relaxed state is associated with breaking of two salt bridges and transient asymmetry of the apical domains (Fei et al, 2013). Asymmetries can be also revealed in the octameric rings of TRiC, an eukaryotic group II chaperonin; in its ATP-binding hierarchy; and even in its transition states (Cong et al, 2012;Reissmann et al, 2012).…”