Hydrophilic Residues 526KNDAAD531 in the Flexible C-terminal Region of the Chaperonin GroEL Are Critical for Substrate Protein Folding within the Central Cavity

Abstract

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region 526 The chaperonin GroEL (14-mer) from Escherichia coli binds denatured proteins and facilitates their folding in vivo and in vitro by encapsulating them within an isolated cavity formed in cooperation with the co-chaperonin GroES (7-mer) (1, 2). Encapsulation by GroEL protects the denatured proteins from interactions with other misfolded proteins or aggregation prone species, providing the proper environment in which the denatured protein may fold spontaneously (3-6). The unique quaternary structure (two heptameric rings stacked back to back) of GroEL enables a clever mechanism. The subunit structure (548 amino acid residues) is divided into three domains; the apical domain, the intermediate domain, and the equatorial domain. Each domain has a specific role in the chaperonin mechanism. The apical domain plays an important role in recognizing and binding denatured protein and the co-chaperonin GroES. The intermediate domain connects the apical and the equatorial domains, and the equatorial domain binds ATP and hydrolyzes it. This ATP hydrolysis controls the overall chaperonin mechanism, regulating binding and release of the substrate protein and GroES (7-11). The refolding substrate protein is encapsulated by GroEL-GroES and segregated from the surrounding environment and, under these conditions, folds correctly without forming aggregation.Thus, the mechanism of GroEL-mediated protein folding is well characterized. However, some details regarding the specific roles of various structural elements in the GroEL subunit structure remain unclear. For example, as shown in Fig. 1, the final 23 amino acid residues of the C terminus are not clearly defined in x-ray crystallographic studies (9) due to high flexibility. However, these segments of GroEL oligomer appear to coalesce and block the central channel at the level of the equatorial domain in electron micrograph (12) and small-angle neutron scattering (13) experiments. Previously, it was reported that this C-terminal region is closely involved in the rate of ATP hydrolysis (14) and GroEL oligomerization (15, 16). However, a mutant with 27-amino acid residues truncated from the C terminus was still functional and could support normal growth of the host cell (15,16). Moreover, the role of the C-terminal region in the mitochondrial homologue Hsp60 (572 amino acid residues) from Saccharo...

Triggering Protein Folding within the GroEL-GroES Complex

“…If these structural changes are important to the allosteric control of GroEL's reaction cycle, it seems reasonable that exogenous dye modification at position 43 could slow or inhibit aspects of this conformational transition. Indeed, several different GroEL variants with modifications of the C-terminal flexible tails, which localize to a similar part of the GroEL cavity, also induce substantial perturbations in assisted protein folding and ATP turnover (35)(36)(37). In the end, a more detailed structural analysis will be necessary to fully address how modifications at position 43 result in the altered folding and ATPase behavior we observe.…”

“…We reasoned that a modified chaperonin like EL43Py might represent a powerful new tool for studying the mechanism of GroELmediated protein folding, especially given the ongoing debate about the importance of the GroEL C-terminal tails, which are located in a similar region at the base of the GroEL cavity (35)(36)(37). To establish the utility of EL43Py, we therefore examined whether the folding deficiency of EL43Py can be explained by a trivial and inhibitory interaction between exogenous dyes localized at the bottom of the GroEL cavity and non-native folding intermediates.…”

The C-terminal Tails of the Bacterial Chaperonin GroEL Stimulate Protein Folding by Directly Altering the Conformation of a Substrate Protein

“…Deletion of these C-terminal tails, which project upward from the equatorial plane of the GroEL ring into the bottom of the cavity, has no effect on the stability of the GroEL tetradecamer but has a notable impact of the folding of several proteins, as well as a modest in vivo phenotype (39,40,(43)(44)(45). Additionally, although the C-terminal tails are not resolved in the x-ray crystal structures of the chaperonin, using high resolution cryo-EM we recently demonstrated that the C-terminal tails interact directly with the non-native Rubisco folding intermediate during and immediately after encapsulation by GroES (46).…”

A Mutant Chaperonin That Is Functional at Lower Temperatures Enables Hyperthermophilic Archaea To Grow under Cold-Stress Conditions

“…McLennan et al found that although the 16 C-terminal residues of GroEL were dispensable for E. coli cell growth, the truncated protein could not complement the groEL(Ts) mutation in E. coli (13). Machida et al reported that the 23 C-terminal residues of GroEL (which are hydrophilic) were not required for chaperonin function (10). However, when the last 6 residues (526-KNDAAD-531) were replaced by a neutral (neither hydrophobic nor hydrophilic) sequence (526-GGGAAG-531) or a hydrophobic sequence (526-IGIAAI-531), chaperonin function was defective both in vitro and in vivo.…”

“…Such coding tandem repeats are usually involved in protein-protein interactions (7). For example, in Escherichia coli, the C-terminal region of GroEL (which is also rich in GGM motifs) is involved in ATP hydrolysis and protein binding (8)(9)(10)(11)(12). Langer et al found that removing the 50 C-terminal amino acid residues of GroEL resulted in a loss of ATPase activity (9).…”