Allosteric mechanisms can be distinguished using structural mass spectrometry

Abstract

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...

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

“…Recent reports include membrane protein complexes (solubilized in detergent micelles) (62)(63)(64), which are particularly challenging for many techniques in structural biology, as well as 18-MDa virus capsids (51), which are thought to be the biggest molecules accessible to the technique with the current state of instrumentation. The resolving power of the technique is often sufficient to monitor posttranslational modifications (such as glycosylation and phosphorylation) and small ligand binding on intact protein complexes up to the megadalton range (18,58,61). Data acquisition takes on the order of minutes between samples, allowing kinetic measurements of relatively slow reactions in the minutes-to-hours timescale (65).…”

“…Ammonium bicarbonate has a higher buffer capacity but may cause protein unfolding at the gas-water interface introduced by bubble formation of the CO 2 gas (17). EDDA was recently introduced and shown to be particularly suitable for the analysis of the ATP-binding GroEL chaperone, resulting in very good desolvation and high-mass resolving power (18).…”

Putting handcuffs on the chaperonin GroEL

“…1) and their biological function. These conformational changes can take place in a concerted fashion according to the MonodWyman-Changeux (MWC) model (2), as proposed and recently demonstrated in the case of the chaperonin GroEL (3,4). Alternatively, the changes can occur in a domino-like sequential fashion according to the Koshland-Némethy-Filmer model (5) or in a probabilistic manner, as suggested in the case of the proteolytic machine ClpP (6).…”

Asymmetric perturbations of signalling oligomers

“…No requirement for crystals, low sample need (pmol) and short measurement time (min) allowing high-throughput processing are considered to be the main strengths of MS. As it has been shown recently, nanoflow ESI-MS studies can distinguish coexisting protein populations with different number of bound ligands (nucleotides for ATPases) and determine the rank order of binding affinities and the role of transient asymmetries of higher-order ring systems in the function of chaperonins and transcription activators (Dyachenko et al, 2013;Zhang et al, 2014).…”