GroEL Allostery Illuminated by a Relationship between the Hill Coefficient and the MWC Model.

A fundamental problem that has hindered the use of the classic Monod-Wyman-Changuex (MWC) allosteric model since its introduction is that it has been difficult to determine the values of its parameters in a reliable manner because they are correlated with each other and sensitive to the data-fitting method. Consequently, experimental data are often fitted to the Hill equation, which provides a measure of cooperativity but no insights into its origin. In this work, we derived a general relationship between the value of the Hill coefficient and the parameters of the MWC model. It is shown that this relationship can be used to select the best estimate of the true combination of the MWC parameter values from all the possible ones found to fit the data. Here, this approach was applied to fits to the MWC model of curves of the fraction of GroEL molecules in the high-affinity (R) state for ATP as a function of ATP concentration. Such curves were collected at different temperatures, thereby providing insight into the hydrophobic effect associated with the ATP-promoted allosteric switch of GroEL. More generally, the relationship derived here should facilitate future thermodynamic analysis of other MWC-type allosteric systems.

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

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.

Allosteric Mechanisms in Chaperonin Machines.

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.

Allosteric mechanisms can be distinguished using structural mass spectrometry.

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 MS--which 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.

Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis.

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 high-throughput 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.