Allostery and molecular machines

09:05-09:30 The nicotinic acetylcholine receptor a typical model of allosteric membrane protein

Professor Jean-Pierre Changeux, Collège de France and Institut Pasteur, France

Abstract

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.

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09:40-10:05 Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor

Professor Vincent J. Hilser, Johns Hopkins University, USA

Abstract

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.

10:35-11:05 New approaches for elucidating allosteric mechanisms and their application to chaperonins

Professor Amnon Horovitz, Weizmann Institute of Science, Israel

Abstract

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.

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11:15-11:45 Cooperative Dynamics of Neurotransmitter Transporters: Learning from Experiments and Computations

Professor Ivet Bahar, University of Pittsburgh, USA

Abstract

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.

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11:55-12:25 Kinetics and thermodynamics of protein assembly

Professor Birgit Strodel, Jülich Research Centre, Germany

Abstract

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.

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13:30-14:00 Breaking symmetry via helix dipoles

Professor George Lorimer FRS, University of Maryland, USA

Abstract

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.

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14:15-14:45 Dynamic GroEL-GroES interaction revealed by high-speed atomic force microscopy

Professor Toshio Ando, Kanazawa University, Japan

Abstract

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.

15:30-16:00 Division of labour among the subunits of a highly coordinated ring ATPase

Professor Carlos Bustamante, University of California, Berkeley, USA

Abstract

As part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. Bacteriophage phi29 packages its 6.6 mm long double-stranded DNA using a pentameric ring nano motor that belongs to the ASCE (Additional Strand, Conserved E) superfamily of ATPases. A number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. The portal motor in bacteriophage phi29 is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and DNA bending energies to package its genome to near-crystalline density inside the capsid. Using optical tweezers, the Bustamante group finds that this motor can work against loads of up to ~55 picoNewtons on average, making it one of the strongest molecular motors ever reported. The group establishes the force-velocity relationship of the motor. Interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to DNA compression attaining the value of ~6 MegaPascals at the end of the packaging. This pressure, Bustamante shows, is used as part of the mechanism of DNA injection in the next infection cycle. The group used high-resolution optical tweezers to characterize the steps and intersubunit coordination of the pentameric ring ATPase responsible for DNA packaging in bacteriophage Phi29. By using non-hydrolyzable ATP analogs and stabilizers of the ADP bound to the motor, they establish where DNA binding, hydrolysis, and phosphate and ADP release occur relative to translocation. Bustamante shows that while only 4 of the subunits translocate DNA, all 5 bind and hydrolyze ATP, suggesting that the fifth subunit fulfills a regulatory function. Finally, he shows that the motor not only can generate force but also torque. The group characterizes the role played by the special subunit in this process and identify this the symmetry-breaking mechanism. These results represent the most complete studies done to date on these widely distribute class of ring nano motors.

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16:15-16:45 Symmetry, rigidity, and Allosteric Wiring Diagram in multi-domain proteins

Professor Dave Thirumalai, University of Texas, USA

Abstract

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.

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09:00-09:30 Allostery in Hsp70s: Intra-molecular pathways and domain dynamics

Professor Matthias Mayer, Heidelberg University, Germany

Abstract

The 70 kDa heat shock proteins (Hsp70s) constitute the central hub of the cellular protein quality surveillance network. The versatility of Hsp70 chaperones is based on the interaction of their substrate binding domain (SBD) with short degenerative sequence motifs, found in most proteins on average every 30 to 40 residues. This binding is controlled by ATP binding and hydrolysis in the nucleotide binding domain (NBD) of Hsp70s, through an intricate allosteric mechanism. ATP binding increases polypeptide substrate association and dissociation rates by 100 and 1000-fold, respectively. In return, polypeptide substrate binding to Hsp70s stimulates the ATP hydrolysis rate in synergism with J-domain cochaperones. This synergistic stimulation of the ATP hydrolysis rate allows Hsp70s to efficiently trap substrates and is essential for all Hsp70 functions.

I will report on extensive structure-function analysis and mutagenesis studies that delineate the allosteric mechanism of Hsp70s, elucidating signal transmission from NBD to SBD. In addition, data on the conformational dynamics of Hsp70s will be presented. Furthermore, we recently solved the crystal structure of the J-domain of E. coli DnaJ in complex with the E. coli Hsp70 DnaK in the ATP bound conformation. Our structure is consistent with a large body of biochemical and biophysical evidence and reveals the molecular mechanism of J-domain protein-mediated stimulation of the ATPase activity of Hsp70s.

09:45-10:15 Can conformational dynamics and allostery be underlying mechanistic principle behind protein evolution?

Professor S. Banu Ozkan, Arizona State University, USA

Abstract

The critical role of protein dynamics has become well recognized in various biological functions, including allosteric signaling and protein ligand recognition electron transfer. Likewise, in protein evolution, the classical view of the one sequence-one structure-one function paradigm is now being extended to a new view: an ensemble of conformations in equilibrium that can evolve new functions. Therefore, understanding inherent structural dynamics are crucial to obtain a more complete picture of protein evolution. A small local structural change due to a single mutation can lead to a large difference in conformational dynamics, even at quite distant residues due to allostery.  We have recently analyzed  evolution of different protein families including GFP proteins, beta-lactamase inhibitors, and nuclear receptors and observed that alteration of conformational dynamics through allosteric regulations leads to functional changes. Moreover, our site-specific dynamics-based metric reveals that enzymatic function is regulated by dynamically-coupled residues, which forms an allosteric communication network with the active sites. Disease causing mutations trigger a global loss in dynamic coupling, which disrupts the communication network ultimately inhibiting function. Analysis of over 200  missense mutations also shows that  Gaucher disease (GD) mutations are abundant at dynamic allosteric residue coupling sites which we call  DARC spots. Further tests using 75 human enzymes revealed that diseases emerging from DARC spot mutations are not isolated to GD; indeed, this phenomenon is observed across the proteome.

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11:00-11:30 Cryo-EM as a tool to explore the proteasome, its function and its ligands

Dr Paula da Fonseca, MRC Laboratory of Molecular Biology, UK

Abstract

The proteasome is essential in all eukaryotes for the highly regulated ATP dependent proteolysis of ubiquitin-tagged proteins, including key cell regulators. In eukaryotes its proteolytic core is formed by four hetero-heptameric rings arranged as an α(1 7)β(1 7)β(1 7)α(1 7) barrel shaped stack, which activity requires the binding of regulators at the outer surfaces of the α subunits. The 19S regulatory particle is the proteasome activator that recruits fully folded ubiquitinated proteins for degradation, associates with the core to form the fully active 26S proteasome and comprises a remarkable variety of functions, including ubiquitin receptors, deubiquitinases, ATP dependent unfoldase and translocase, and scaffolding. These are accomplished by at least 18 distinct canonical subunits, together with additional transiently bound proteins that further modulate the proteasome activity. Recently there was substantial progress in determining the 26S proteasome structure. However, a complete characterisation of how the proteasome components orchestrate the individual steps of substrate processing, from their recognition to the release of small peptides, is still missing. This information is required to fully understand the proteasome fundamental role and in order to optimise specific ligands as therapeutic drugs. In principle the final stages of substrate processing can be studied by the structural analysis of the proteasome core in the presence of substrate or product peptide mimetics. We investigated the use of cryo-EM and image processing for such studies, revealing advantages of their use for the overall study of protein-ligand interactions and leading to our contribution to the validation of the proteasome as a potential target for antimalarials.

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11:45-12:15 Slow Brownian fluctuations for allosteric signalling without structural change

Professor Tom McLeish FRS, Durham University, UK

Abstract

McLeish presents the results of a physics-biology collaboration in a foundational theory for how allostery can occur as a function of low frequency dynamics without a change in protein structure. Elastic inhomogeneities allow entropic ‘signalling at a distance’. Remarkably, many globular proteins display just this class of elastic structure, in particular those that support allosteric binding of substrates (long-range co-operative effects between the binding sites of small molecules). Through multi-scale modelling of global normal modes McLeish demonstrates negative co-operativity between the two cAMP ligands without change to the mean structure. Crucially, the value of the co-operativity is itself controlled by the interactions around a set of third allosteric ‘control sites’. The theory makes key experimental predictions, validated by analysis of variant proteins by a combination of structural biology and isothermal calorimetry. A quantitative description of allostery as a free energy landscape revealed a protein ‘design space’ that identified the key inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, by analysing naturally occurring CAP variants from diverse species, McLeish demonstrates an evolutionary selection pressure to conserve residues crucial for allosteric control. The methodology establishes the means to engineer allosteric mechanisms that are driven by low frequency dynamics.

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13:30-14:00 Time-resolved modelling of protein allosteric communication

Professor Gerhard Stock, University of Freiburg, Germany

Abstract

Allostery represents a fundamental mechanism of biological regulation which is mediated via long-range communication between distant protein sites. While little is known about the underlying dynamical process, recent time-resolved infrared spectroscopy experiments on a photoswitchable PDZ domain (PDZ2S) have indicated that the allosteric transition occurs on multiple timescales. Employing extensive nonequilibrium molecular dynamics simulations, here a time-dependent picure of the allosteric communication in PDZ2S is developed. The simulations reveal that allostery amounts to the propagation of structural and dynamical changes that are genuinely nonlinear and can occur in a nonlocal fashion. A dynamic network model is constructed that illustrates the hierarchy and exceeding structural heterogeneity of the process. In compelling agreement with experiment, three physically distinct phases of the time evolution are identified, describing elastic response ( 0.1 ns), inelastic reorganization (∼ 100 ns) and structural relaxation ( 1μs). Issues such as the similarity to downhill folding as well as the interpretation of allosteric pathways are discussed. 

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14:15-14:45 Exploring cooperativity of folding and binding in the tandem-repeat protein class

Dr Laura Itzhaki, University of Cambridge, UK

Abstract

I will discuss the cooperative nature of protein folding and its relevance for function in the context of two contrasting systems. The first is the cks family of cell-cycle regulatory proteins. These proteins undergo three-dimensional domain swapping, in which two monomers exchange a part of their structure to form a highly intertwined dimer. Our work has explored how features of the cks architecture that are essential for domain swapping also control signal transduction between multiple binding partners that facilitate cks function as a hub for macromolecular complex assembly. The second is the tandem-repeat protein class such as ankyrin and tetratricopeptide repeats. Repeat proteins have strikingly simple, modular structures composed of small (30-50 residue) motifs repeated multiple times in tandem to produce elongated pseudo-one-dimensional spring-like architectures. We have been investigating how the folding cooperativity of repeat proteins can be manipulated by the insertion of long unstructured loops between adjacent repeats. In Nature such insertions, and the decoupling of the repeats that results, are likely to be important for the mechanics and the biological functions of these proteins.

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15:30-16:00 Evolution of specificity for allosteric regulators: mechanisms and constraints

Professor Joseph Thornton, University of Chicago, USA

Abstract

We have used ancestral protein reconstruction to identify the structural and genetic mechanisms by which specificity for allosteric effectors evolved in the steroid hormone receptor family of ligand-activated transcription factors.   We find that dramatic shifts in ligand specificity -- and even a wholesale loss of allosteric control -- occurred numerous times during receptor evolution and were often mediated by a few mutations of large effect.  The delicate energetics of allosteric regulation imposed strong constraints on the proteins' evolution, requiring rare permissive mutations to stabilize specific structural elements before changes in ligand regulation could occur.