The activity of many proteins, including metabolic enzymes, molecular machines, and ion channels, is often regulated by conformational changes that are induced or stabilized by ligand binding. In cases of multimeric proteins, such allosteric regulation has often been described by the concerted Monod-Wyman-Changeux and sequential Koshland-Némethy-Filmer classic models of cooperativity. Despite the important functional implications of the mechanism of cooperativity, it has been impossible in many cases to distinguish between these various allosteric models using ensemble measurements of ligand binding in bulk protein solutions. Here, we demonstrate that structural MS offers a way to break this impasse by providing the full distribution of ligand-bound states of a protein complex. Given this distribution, it is possible to determine all the binding constants of a ligand to a highly multimeric cooperative system, and thereby infer its allosteric mechanism. Our approach to the dissection of allosteric mechanisms relies on advances in MSwhich provide the required resolution of ligand-bound states-and in data analysis. We validated our approach using the well-characterized Escherichia coli chaperone GroEL, a double-heptameric ring containing 14 ATP binding sites, which has become a paradigm for molecular machines. The values of the 14 binding constants of ATP to GroEL were determined, and the ATP-loading pathway of the chaperone was characterized. The methodology and analyses presented here are directly applicable to numerous other cooperative systems and are therefore expected to promote further research on allosteric systems.chaperonins | Hill coefficient M ultimeric proteins are often subject to allosteric regulation that is achieved by conformational changes induced or stabilized by ligand binding (1). Such allosteric regulation has been described by two classic models: (i) the Monod-WymanChangeux (MWC) model (2), in which conformational changes occur in a concerted manner and symmetry is conserved, and (ii) the Koshland-Némethy-Filmer (KNF) model (3), in which conformational changes take place in a sequential manner and symmetry is broken. In addition, it has been proposed more recently that conformational changes can take place in a probabilistic manner (4). The allosteric control of protein activity is frequently manifested in sigmoidal plots of initial reaction velocity or fractional saturation as a function of the ligand (substrate) concentration that indicates positive cooperativity in ligand binding. It has been impossible, however, to extract any mechanistic insights from these plots (5) because they only show how an average property of the ensemble (e.g., fractional saturation) changes with ligand concentration and do not reveal how the distribution of ligand-bound states changes with ligand concentration. Thus, for example, it is not possible to determine from such sigmoidal plots whether an allosteric transition takes place in a concerted MWC-like fashion (2) or via a sequential KNF-like mechanism (3). T...

Chaperonins are nanomachines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space owing to complex allosteric regulation. They consist of two back-to-back stacked oligomeric rings with a cavity at each end where protein substrate folding can take place. Here, we focus on the GroEL/GroES chaperonin system from Escherichia coli and, to a lesser extent, on the more poorly characterized eukaryotic chaperonin CCT/TRiC. We describe their various functional (allosteric) states and how they are affected by substrates and allosteric effectors that include ATP, ADP, nonfolded protein substrates, potassium ions, and GroES (in the case of GroEL). We also discuss the pathways of intra- and inter-ring allosteric communication by which they interconvert and the coupling between allosteric transitions and protein folding reactions.

The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used highthroughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems. molecular chaperones | polyQ proteins | protein mis-folding | protein aggregation | high-content analysis C haperonins are ATP-dependent protein-folding machines that are present in all kingdoms of life. They consist of two back-toback stacked oligomeric rings with a cavity at each end, where protein substrate binding and folding take place (for reviews see refs. 1 and 2). The chaperonins can be divided into two groups: group I, found in the bacterial cytoplasm (e.g., GroEL in Escherichia coli), mitochondria, and chloroplasts; and group II found in archaea and the eukaryotic cytosol. Numerous studies have shown that the group I chaperonin GroEL can facilitate the folding of a large number of different proteins in vitro, although its role in the cell is much more limited (for review see ref.3). The group II eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/ TRiC) also seems to have a specialized role in vivo in the folding of actin (4), tubulin (5), and other essential proteins, including regulators of cell division and cytoskeleton formation (6-8), despite seeming to possess broad binding specificity (6). The list of members in the CCT interactome is, however, not yet fully established, and the conditions for entry into this exclusive club remain poorly understood.An important distinction between group I and group II chaperonins is that the former consist of homo-oligomeric rings, whereas the latter usually consist of hetero-oligomeric rings that contain two or three different subunits in the case of many archaeal chaperonins and eight different subunits in the case of CCT. The eight subunits of CCT are arranged in a defined permutation (9), and the orientation of the two rings of CCT with respect to each other is also fixed (10). CCT's heter...

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...

Advances in native mass spectrometry and single-molecule techniques have made it possible in recent years to determine the values of successive ligand binding constants for large multi-subunit proteins. Given these values, it is possible to distinguish between different allosteric mechanisms and, thus, obtain insights into how various bio-molecular machines work. Here, we describe for ring-shaped homo-oligomers, in particular, how the relationship between the values of successive ligand binding constants is diagnostic for concerted, sequential and probabilistic allosteric mechanisms.This article is part of a discussion meeting issue 'Allostery and molecular machines'.