Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis

Abstract

Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential "conformational waves." They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively.chaperonins | allostery | conformational changes | molecular machines A TP-fueled ring-shaped nanomachines are ubiquitous and found in all domains of life. Examples for such machines include chaperones that mediate protein folding and degradation (1), DNA and RNA remodeling enzymes (2), and proteins involved in intracellular trafficking (3). The oligomeric rings that form these machines usually consist of five to eight subunits that undergo coordinated conformational changes driven by ATP binding and/or hydrolysis. These conformational changes can take place in a concerted fashion as in the case of the chaperonin GroEL (4). Alternatively, they can also occur in a sequential or stochastic manner as reported, for example, for the CCT/TRiC chaperone complex (4) and ClpX (5), respectively. Knowing the mode of allosteric switching is essential for understanding the mechanism of action of biomolecular machines. Distinguishing between these different modes of allosteric switching can be achieved using native mass spectrometry (6, 7) and single-molecule fluorescence (8) and force-based (2) techniques but has been difficult to accomplish using traditional biochemical approaches. Here, we show that classical Arrhenius analysis can be used to unpick the allosteric mechanism of ATP hydrolysis by the CCT/ TRiC chaperone.CCT/TRiC is a member of group II chaperonins found in archaea and the eukaryotic cytosol that assist protein folding in an ATPdependent manner. Clients of this chaperonin system include β-actin, α-and β-tubulin, and several hundred other proteins (9, 10). CCT/TRiC consists of two identical back-to-back stacked octameric rings with a cavity at each end where protein folding can take place (11). Each ring of CCT/TRiC is made up of eight different subunits that are arranged in a fixed order around the ring. The correct order was established by determining which arrangement is most consistent with interresidue distance constraints obtained from chemical crosslinking and mass spectrometry (12). This order was also found to give the best fit to the crystallographic data for CCT/TRiC (9) and was, therefore, used to redetermine its structure accordingly (13). The eight subunits of CCT/TRiC have a similar structure that consists of three domains: (i) an apical domain that is involved in protein substrate binding, (ii) an equatorial domain that is involved in ring-ring interactions and contains an ATP binding site, and (iii) an intermediate domain that l...

Structural basis for active single and double ring complexes in human mitochondrial Hsp60-Hsp10 chaperonin

“…β-barrel mobility is diminished compared to mHsp60 apical domain sections. We conclude that the subunit conformational asymmetry observed for the ground state football may represent snapshots of different dynamic subunit conformations, which are consistent with the B-factor analysis and provide a basis for an intra-ring communication mechanism akin to that observed for CCT 38 .…”

Intraring allostery controls the function and assembly of a hetero‐oligomeric class II chaperonin

“…The same phenomenon, but of opposite sign, has been observed in CCT from S. cerevisiae , inwhichATP‐hydrolysis mutants of the strong (β ‐equivalent) subunits were found to affect cell viability the most (14, 15). This was interpreted as a way to connect, through subunit‐substrate (or substrate domain) specificity, the ATP‐cycling trajectory to a specific folding pathway (17).…”

“…This has been confirmed by cryo‐EM analysis of a newly identified state, partially preloaded with nucleotide, and of the ATP‐bound state (16). Further analysis of the data on ATP‐site mutants led to the proposal of a model describing an intraring sequential allosteric mechanism of ATP hydrolysis by CCT (17). It has been speculated, based on the differential characteristics and behavior of the subunits, that such a complex mechanism could increase the efficiency of folding of multidomain proteins by allowing their release from the chaperonin, domain by domain, in a sequential order (15, 17‐19).…”

The substrate specificity of eukaryotic cytosolic chaperonin CCT

“…The selective nature of the CCT machinery and its individual subunits was revealed in cryo-EM maps of CCT-actin [9] and CCT-tubulin complexes [10,11] and through direct biochemical analyses of actin binding sites [12 -14]. CCT shows sequential allosteric behaviour in its ATP-binding and hydrolysis regime [15,16] and in its actin-folding regime [4, 11,17]. Our long-standing view is that CCT actively folds a very restricted group of non-cytoskeletal proteins which includes many members of the 7-bladed WD40-propeller repeat protein family.…”