Allostery and molecular machines

The concept of allosteric interaction (1) which was initially proposed to account for the inhibitory feedback mechanism mediated by bacterial regulatory enzymes contrasts with the classical mechanism of competitive, steric, interaction between ligands for a common site. Accordingly allosteric interactions are indirect interactions that take place between topographically distinct sites and are mediated by a discrete & reversible conformational change of the protein. 
The concept was soon extended to membrane receptors for neurotransmitters (2) which behave as «molecular switches»  mediating the signal tranduction process at the synapse, which, in the case of the acetylcholine nicotinic receptor (nAChR), links the ACh binding site to the ion channel (3). Furthermore, pharmacological effectors, referred to as allosteric modulators, such as Ca++ ions and ivermectin, were discovered that enhance the transduction process when they bind to sites distinct from the orthosteric ACh site and the ion channel on the nAChR (4).  The recent X-ray structures, at atomic resolution, of the resting & active conformations of prokaryotic and eukaryotic homologs of the nAChR, in combination with atomistic molecular dynamics simulations (5) reveal a stepwise  quaternary transitions in the transduction process with tertiary changes which modify the boundaries between subunits. These interfaces host orthosteric and allosteric modulatory sites which structural organization changes in the course of the transition. The model emerging from these studies has lead to the conception and development of new pharmacological agents. For example, looking for chemical therapies against Autism, a strategy was  elaborated on the basis of brain genes expression data, using the concept of coherent-gene groups controlled by transcription factors (TFs), which resulted in the design of allosteric modulators targeted toward specific TFs expressed at critical steps of brain development (6). 

1. Changeux JP (1961) The feedback control mechanisms of biosynthetic L- threonine deaminase by L-isoleucine. Cold Spring Harb Symp Quant Biol 26:313–318 ; Gerhart JC, Pardee AB (1962] The enzymology of control by feedback inhibition. J Biol Chem 237:891–896
2. Changeux (1964) PhD Thesis ; (1965) [On the allosteric properties of biosynthesized l-threonine deaminase. VI. General discussion]. Bull Soc Chim Biol (Paris) 47:281-300.
3. Taly A, Corringer PJ, Guedin D, Lestage P, Changeux JP. (2009) Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system Nat Rev Drug Discov. 8:733-50. 
4. Corringer, P.J., Poitevin, F., Prevost, M.S., Sauguet, L., Delarue, M., Changeux, J.P.(2012) Structure and pharmacology of pentameric receptor-channels: from bacteria to brain. Structure 20, 941–956 
5. Changeux JP (2014) Protein dynamics and the allosteric transitions of pentameric receptor channels. Biophys Rev. 6:311-321 ; Cecchini M, Changeux JP (2014) The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation. Neuropharmacology Dec 18. pii: S0028-3908(14)00450-X. doi: 10.1016/j.neuropharm.2014.12.006
6. Tsigelny IF, Kouznetsova VL, Baitaluk M, Changeux JP.(2013) A hierarchical coherent-gene-group model for brain development. Genes Brain Behav. 12:147-65.

Intrinsically disordered proteins (IDPs) present a functional paradox because they lack stable tertiary structure, but nonetheless play a central role in signaling, utilizing a process known as allostery. Historically, allostery in structured proteins has been interpreted in terms of propagated structural changes that are induced by effector binding. Thus, it is not clear how IDPs, lacking such well-defined structures, can allosterically affect function. Here we show a mechanism by which an IDP can allosterically control function by simultaneously tuning transcriptional activation and repression, using a novel strategy that relies on the principle of energetic ‘frustration’. We demonstrate that human glucocorticoid receptor tunes this signaling in vivo by producing translational isoforms differing only in the length of the disordered region, which modulates the degree of frustration. We expect this frustration-based model of allostery will prove to be generally important in explaining signaling in other IDPs.

Chaperonins are allosteric machines that consist of two back-to-back stacked heptameric rings with a cavity at each end where protein folding can take place.  They assist protein folding by undergoing large conformational changes that are controlled by ATP binding and hydrolysis. The concerted Monod–Wyman–Changeux and sequential Koshland–Némethy–Filmer models of cooperativity are often used to describe such allosteric switching.  In general, however, it has been impossible to distinguish between these different allosteric models using ensemble measurements of ligand binding in bulk protein solutions.  In this talk, two approaches that break this impasse will be described: one that is kinetic and a second that is based on native mass spectrometry.  Using these approaches, it was possible to show that the chaperonin GroEL from E. coli undergoes concerted intra-ring conformational changes whereas its eukaryotic homologue CCT/TRiC undergoes sequential intra-ring conformational changes.  The impact of these different allosteric mechanisms on the folding functions of GroEL and CCT/TRiC will be discussed.

Recent years have seen a breakthrough in the elucidation of the structure and dynamics of sodium-coupled neurotransmitter transporters. These membrane proteins are essential regulators of neurotransmission in the brain, and their malfunction is implicated in several neurological disorders. We have now made significant progress in understanding the complex machinery of these secondary transporters, the way they undergo cooperative structural changes between outward-facing and inward-facing states for transporting their substrate and sodium ions, while they also permit for chloride channeling. We will present recent progress made in the elucidation of the mechanism of function of two major groups of transporters and their alteration by ligand binding and/or multimerization: Glutamate transporters, exemplified by the archaeal transporter GltPh which served as a useful model for understanding the dynamics of excitatory amino acid transporters (EAATs); and dopamine transporters as an important member of transporters sharing the LeuT fold. We will show how the multidomain structure or multimerization properties are essential to altering not only their conformational dynamics, but also the coupled membrane remodeling in the synapse, based on recent progresses made in both visualizing and modeling the molecular-to-cellular dynamics of these important transporters that regulate glutamatergic and dopaminergic signaling in the central nervous system.

The aim of my work is to understand the physicochemical principles that govern the highly complex process of protein assembly. This process may lead to fatal diseases, as in the case of Alzheimer's disease, but life also profits from it as many molecular processes within a cell are carried out by molecular machines that are built from a large number of proteins. All-atom molecular dynamics (MD) simulations of protein assembly in explicit solvent have been performed for over a decade, revealing valuable information about this phenomenon. The focus of my work lies on the analysis of MD simulations to elucidate the kinetics and thermodynamics of protein assembly processes. To this end, we developed kinetic transition networks showing the transitions between aggregates of different sizes and structural characteristics, allowing us to extract both the thermodynamics and kinetics of the assembly process. While the kinetic transition networks are based on conformational clustering, Markov state models (MSMs) use kinetic clustering for the identification of metastable states. The application of MSMs to protein assembly is highly desirable but challenging, as I will demonstrate in this talk for the aggregation of a small peptide. I will conclude my talk with a perspective on how the methods developed in my group can be applied to molecular machines in order to identify structural changes and kinetically relevant intermediates in their functional cycle.

The dipole moment of -helices (positive at the N-terminus, negative at the C-terminus is an important property that is rarely exploited in the transmission of allosteric information.  Evidence will be presented here that the nucleotide binding sites of the different rings of the chaperonin GroEL communicate via the dipole moment of the 21-residue helix D (Gly88- Ala109).  In the apo-state of GroEL the -amino group of Lys105 of helix D of one ring forms an inter-ring salt bridge with the negatively charged carbonyl oxygen of Ala109 of helix D of the other ring.  Upon binding ATP the -phosphate interacts with the positively charged Gly88 at the N-terminal end of helix D, causing a ~2Å rigid body shift of the entire helix. This results in the breakage of the inert-ring Lys105-Ala109 salt bridges that are responsible for the negative cooperativity in ATP binding between the rings.  The mutant K105A (i) removes these inter-ring salt bridges, (ii) abolishes negative cooperativity in ATP hydrolysis between the rings without altering the positive cooperativity within a ring or the fundamental rate of ATP hydrolysis determined from the pre-steady state burst kinetics, (iii) compromises the ability of the symmetric GroELK105A-GroES2 “football” complex to undergo breakage of symmetry. Absent the electron density associated with the side chains of K105, the crystal structure of apo-GroELK105A is virtually identical to that of apo-GroELWT.

The GroEL−GroES chaperonin reaction system contains two rings of GroEL, seven ATPase sites in each ring, co-chaperonin GroES and substrate protein. Therefore, the reaction cycle of this system is too complicated to be analyzed by ensemble methods. Single-molecule fluorescence microscopy studies were recently performed and revealed a part of the reaction scheme with major appearance of the symmetric GroEL−(GroES)2 complexes. Nevertheless, the two rings of GroEL are closely positioned, so that it was difficult to know from which ring a detected fluorescence signal came. Here we used high-speed atomic force microscopy to visualize the dynamic GroEL−GroES interaction in the presence of ATP and unfoldable substrate protein, disulfide bond-reduced α-lactalbumin. The GroEL−GroES interaction was observed to branch into main and side pathways with probabilities of 2/3 and 1/3, respectively. In the main pathway, alternate binding and release of GroES occurs at the two rings. This alternate rhythm is made possible by two types of inter-ring communications; (i) the completion of ATP hydrolysis to ADP∙Pi at the new cis ring triggers Pi-release (and hence GroES release) from the opposite ring and (ii) the resulting asymmetric bullet-shaped structure retards ADP dissociation from the trans ring. This retardation contributes to providing an enough time for the substrate protein to be released from the trans ring but in turn possibly results in incomplete replacement of ADP with ATP at the trans ring. This incompletion is likely to deteriorate the inter-ring communication, resulting in the break of the alternate rhythm.

I will introduce concepts describing the physical basis of allostery relying on propagation of excitations (phonons for example) in ordered solids. These ideas will be used to develop a Structural Perturbation Method (SPM) for identifying a network of residues that carry signals for allosteric transitions. Applications to bacterial chaperonin (GroEL) will be presented. In addition, the connection between the static SPM theory and the dynamics associated with allosteric transitions will be established.