Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins

Department of Molecular and Cellular Physiology, Stanford University, Palo Alto, California, United States
Nature Structural & Molecular Biology (Impact Factor: 13.31). 06/2007; 14(5):432-40. DOI: 10.1038/nsmb1236
Source: PubMed


Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.

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    • "For psychrophilic enzymes, amino acid substitutions that facilitate the flexibility of the hinge regions about which loops or domains must move are the key adaptive changes that lower activation energy barriers for catalytically critical conformational changes. Therefore, we propose that adaptive evolution of CCT for efficient function at psychrophilic temperatures may be based upon flexibility-enhancing residue substitutions in the apical lid, which appears to control the rate-limiting transition from the closed to open state as ADP and Pi are released (Reissmann et al., 2007). We have not examined this step of the catalytic cycle, but our measurements of the temperature dependence of the ATPase activities of apoCCTs strongly supports this hypothesis – the thermal coefficient of G. gibberifrons CCT is consistent with maintenance of structural flexibility at 4°C, whereas that for bovine CCT indicates a loss of flexibility at this temperature. "
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    ABSTRACT: Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT-CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT-CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = -1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between -4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits.
    Biology Open 03/2014; 3(4). DOI:10.1242/bio.20147427 · 2.42 Impact Factor
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    • "To further investigate the reduced ATP hydrolysis rate in Cpn-G160S, we monitored the ATPase activity over a longer time scale (Figure 4C). The ATP hydrolysis rate of Cpn-G160S was compared with the catalytically dead mutant Cpn-D386A, as negative control (Reissmann et al, 2007). These experiments suggested that the Cpn-G160S retains ATPase activity, albeit at a much slower rate than observed in the Cpn-WT. "
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    ABSTRACT: Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.
    The EMBO Journal 12/2011; 31(3):731-40. DOI:10.1038/emboj.2011.468 · 10.43 Impact Factor
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    • "These apical protrusions are unique to group II chaperonins, like thermosome and CCT, and they have been proposed to have GroES-like activity and act as a built-in lid that might open and close in an ATP-dependent manner [17, 85]. Compelling evidence for the requirement of lid responsible for encapsulating the unfolded polypeptides comes from the observations that lidless chaperonins lose the ability to fold stringent substrates [65, 86, 87]. The question is how does this “built-in lid” functions for the closing and opening of the central cavity for substrate binding and releasing? "
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    ABSTRACT: Molecular chaperones are a class of proteins responsible for proper folding of a large number of polypeptides in both prokaryotic and eukaryotic cells. Newly synthesized polypeptides are prone to nonspecific interactions, and many of them make toxic aggregates in absence of chaperones. The eukaryotic chaperonin CCT is a large, multisubunit, cylindrical structure having two identical rings stacked back to back. Each ring is composed of eight different but similar subunits and each subunit has three distinct domains. CCT assists folding of actin, tubulin, and numerous other cellular proteins in an ATP-dependent manner. The catalytic cooperativity of ATP binding/hydrolysis in CCT occurs in a sequential manner different from concerted cooperativity as shown for GroEL. Unlike GroEL, CCT does not have GroES-like cofactor, rather it has a built-in lid structure responsible for closing the central cavity. The CCT complex recognizes its substrates through diverse mechanisms involving hydrophobic or electrostatic interactions. Upstream factors like Hsp70 and Hsp90 also work in a concerted manner to transfer the substrate to CCT. Moreover, prefoldin, phosducin-like proteins, and Bag3 protein interact with CCT and modulate its function for the fine-tuning of protein folding process. Any misregulation of protein folding process leads to the formation of misfolded proteins or toxic aggregates which are linked to multiple pathological disorders.
    07/2011; 2011:843206. DOI:10.4061/2011/843206
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