Biological cells perform their fundamental tasks through the orchestrated action of numerous proteins and cellular assemblies. Large molecular assemblies synthesize natural products, metabolites, and proteins, assist protein folding or unfolding, or allow the communication within cells and with their environment. Understanding how these "nanomachines" work on the atomic scale has stimulated decades of research. A particularly fascinating aspect of the action of such machineries is that they often function through the concerted of different parts of the molecule, often through poorly understood long-range couplings. In this project we focus on the ClpP protease, a 300 kDa large protein assembly which features some of the hallmarks of such allosteric coupling and concerted mechanisms. ClpP is able to degrade proteins which enter the central enzymatic cavity through axial pores, and cooperates with its cognate AAA+ chaperone, which delivers unfolded polypeptide chains to the protease. The caseinolytic protease (Clp) system is a paradigmatic case of a protease machinery, present in bacteria and most eukaryotes. Clp plays an active role in survival and virulence of pathogenic bacteria. Therefore, the development of drugs targeting ClpPs has recently emerged as a promising strategy to address multi-resistant bacteria. Furthermore, indications that mitochondrial ClpP expression is induced in response to cellular stress, e.g. as a consequence of tumor genesis, stresses the importance of ClpP proteases in eukaryotic protein homeostasis. While structures are available, the mechanism of the interaction between ClpP and it co-chaperone and the gate opening of the protease remain unclear. Dysrfunction of the Clp system can cause major physiological defects in bacteria. In particular, acyldepsipeptide (ADEP), a natural peptide-based compound, has been shown to activate ClpP, converting it from a highly-regulated peptidase that can degrade proteins only with the aid of its partner AAA+ to an independent and unregulated protease. ADEP treatment was shown successful to be effective antibiotics for bacteria resistant to other treatments. Furthermore, a novel class of antibiotics is able to activate ClpP, via a not fully resolved allosteric gate opening. Here we integrate information from multiple experimental techniques, centered around solution and magic-angle-spinning NMR, SAXS and EM, with advanced in silico simulation methods to elucidate the mechanisms of conformational exchange and allosteric coupling in ClpP. We investigate the effects that activators or co-chaperones have on ClpP structure and dynamics to gain insight into the mechanisms that exceed the information that any single method alone may obtain. We further address the effect of drugs on the allosteric transition leading to impaired activation of ClpP. In addition to providing mechanistic insights into protease machineries, this project pushes back the frontiers of both NMR – with new site-specific NMR dynamics experiments collected in a system that exceeds by far standard NMR applications – and MD simulations, developing original strategies for enhanced ergodic sampling.

The goal of this project is to use the resurrection of ancestral proteins for investigating the genesis of allosteric regulation. As now accepted, a protein is not a static entity, on the contrary its structure fluctuates, and continuously explores a range of conformations. The relative occupancy of each state as well as their interconversions are controlled by the free energy landscape. The finest details of the conformational landscape depend on the primary sequence of the protein, and therefore are critically altered by amino-acid substitutions as they can occur along the molecular evolution. Determining the evolvability potential of each amino acid replacement, or in other words, determining the magnitude of its impact on the protein conformational landscape, is a key point to clearly understand the evolution of proteins, and of their functions, and most importantly of the mechanism underlying activity regulation. In the project AlloAnc, we will use a synergetic approach based on various experimental and theoretical methods: ancestral sequence reconstruction and protein resurrection together with biochemical, structural and dynamical investigations. Using this approach, we propose to reconstruct the genesis of the allosteric regulation in a superfamily of dehydrogenases. This superfamily is divided into several enzymatic groups: the Lactate dehydrogenases (LDHs), which are in great majority allosteric and the Malate and hydroxyacid dehydrogenases (MalDHs and HincDH) which are not. Allosteric regulation in LDHs is due to the binding of an allosteric effector: the fructose bis-phosphate (FBP) that is an intermediate of the glycolytic cascade. FBP strongly activates catalytic efficiency of allosteric LDH. Since the family divergence, contemporary LDHs, MalDHs and HincDH differ by three main properties: their functions, their capacity of regulation and their conformational flexibility. Our working hypothesis is that the genesis of the allosteric properties in LDHs is the result of the critical effect of mutations cumulated outside the catalytic site. These mutations had long-range effects on the catalytic site, namely by inducing enhanced flexibility would cause its distortion from the functional structure and therefore knocked-out the activity. However, this loss of activity can be viewed as an efficient strategy to ensure its control if other mutations are able to restore the correct catalytic site geometry by promoting, for example, the binding of a ligand which will suppress the un-functional flexibility and ultimately restoring the activity. This is the essential principle of regulation by allosteric activation. In the case of LDHs, the substitutions allowing the binding of the FBP were able to create conditions for a balance between inactive (too flexible) and active conformers (less flexible) within a single molecule. The objective of our project is to trace by phylogenetic approaches the evolutionary pathways that led to modern LDHs and to experimentally characterize the ancestral enzymes occupying the key positions along the divergence pathways and therefore to reconstruct the main steps in the genesis of allostery. Our approach is the only one that allows both a determination of the respective order of fixation of amino acid substitutions and a measurement of the magnitude of their dynamical effects in an evolutionary process. This will allow us to dissect not only the local effects of mutations but also to understand how their long-distance effects propagate. The results obtained will provide major advances not only in fundamentals science but also in the design of therapeutic enzyme inhibitors. Indeed, thanks to high throughput screening of bioactive compounds we will identify “allosteric inhibitor like” molecules and we will analyze the results with respect to the library of dynamical structures obtained by molecular dynamics simulation.

Natural products and their biosynthetic routes are important sources of inspiration for chemists in their search for new environment-friendly and still highly efficient routes for the synthesis of fine chemicals. Carbon-carbon bond formation and cleavage are among the most difficult reactions. They usually require preliminary activation steps through the insertion of several, often transitory, functional groups. This frequently leads to an increase in the number of steps and a concomitant drop in the yields. Radical-based chemistry is a good alternative, thanks to its unique ability to activate otherwise unreactive positions such as aliphatic carbon atoms. The downside of this approach is that radical-based reactions are really difficult to control. In Nature, radical S-adenosyl-L-methionine (rSAM) enzymes are versatile radical catalysts capable of performing and tightly controlling over seventy different chemical reactions. With the CARBONARA project, we aim at determining the factors that control and drive radical-based C-C bond cleavage in rSAM enzymes. We want to address the key issues of substrate selectivity and activation, and the questions as to how these proteins control the radical-based chemistry to cleave one particular C-C bond over another and what tightly controls the termination of the reaction and the final products that are formed. To achieve these goals, we have selected five proteins with similar folds, readily available within the consortium, that catalyze C-C bond cleavage in amino acids. Our consortium consists of three internationally renowned partners with complementary expertise in the fields of biochemistry, structural biology and spectroscopy covering the fundamentals of this fascinating chemistry. Using X-ray crystallography, in vitro functional analyses, electron paramagnetic resonance spectroscopy and theoretical calculations, we want to characterize the functional, structural and electronic parameters that control the C-C cleavage reaction. In this way, we expect to understand and identify the key factors, which are intrinsic to the radical-based reaction itself and the extrinsic ones controlled by the protein matrix. Our original multidisciplinary approach should not only favor a more rational use of radical-based chemistry in organic synthesis, but also lay the foundations for the use of rSAM enzymes as new tools in synthetic biology, in particular through the development of rationally designed chimeras.

A complete description of biomolecular activity requires an understanding of the nature and the role of protein conformational dynamics. In recent years novel nuclear magnetic resonance (NMR) techniques have emerged that provide hitherto inaccessible detail concerning biomolecular motions occurring on physiologically important timescales. In particular residual dipolar couplings (RDCs) provide precise information about time and ensemble averaged structural and dynamic processes with correlations times up to the millisecond, and thereby encode key information for understanding biological activity. In recent years we have developed two very different approaches to the quantitative description of intrinsic protein motions on a wide range of timescales using RDCs. Application of these techniques to the study of the proteins Ubiquitin (Ub) and GB3 resulted in the convergent observation of enhanced dynamic fluctuations occurring on intermediate timescales (nano to millisecond) in the physiological interaction sites of these proteins. The motions occurring in these interaction sites were suggested to exhibit specific modes that would either optimally accommodate the interaction partner, or to intrinsically sample the conformations found in complex with diverse functional partners. In this study we will investigate, for the first time, the nature and timescale of these slower motions, not only in the isolated proteins, but also in the presence of different interaction partners. Concentrating on a specific and important cellular paradigm, we focus on characterizing the interaction between Ub and different Ub binding domains (UBDs). Ub is a versatile cellular signal, regulating a wide variety of activities ranging from protein degradation and quality control, endocytosis, transcriptional regulation to cell signaling and membrane trafficking. Ub has a large number of intracellular partners (more than 150 have been annotated), and while affinities of mono-Ub-binding interactions are very often weak, they span two orders of magnitude (Kd 3-2000µM). We will study the interaction between Ub and a range of UBDs with different affinities in order to probe the possible link between the structural dynamics of the molecular complex occurring on timescales up to the millisecond, and the kinetics of the interaction. In order to achieve this goal we will build on and extend state-of-the-art experimental, analytical, numerical and molecular simulation techniques established over the last ten years in the laboratory of the coordinator to measure and analyse RDCs, spin relaxation and relaxation dispersion from diverse Ub-UBD complexes. During the course of this project we will (a) compare, for the first time, the nature of large scale slow motions in the presence and absence of functional partners, (b) establish the dependence of interaction affinity on molecular recognition dynamics in free and bound forms of interacting partners, (c) extend the timescale over which NMR can be used to determine local entropic and enthalpic contributions to the thermodynamic equilibrium (d) more than double the number of individual proteins whose backbone dynamics have been characterised using RDCs, thereby establishing general trends concerning the nature of slow motions across different protein families. In summary, this project fully exploits the unique sensitivity of NMR to study weak protein-protein interactions at atomic resolution to address a problem of great current importance. While methods have been developed to describe slower motions in proteins, with the observation that these dynamics tend to occur in the interaction site of the proteins, the modulation of this flexibility upon interaction remains unknown. Methods developed in the group of the coordinator over the last decade will be applied in the course of this project to compare protein dynamics in the free and bound forms of Ub and UBDs participating in weak complexes.

Poisoning by organophosphorus compounds (OP) is a serious worldwide public and military health issue. OP nerve agents present a threat in terrorist attacks and conflicts (civil war in Syria). The acute toxicity of OP agents results from their irreversible inhibition of acetylcholinesterase (AChE), which regulates cholinergic transmission in the peripheral and centralnervous system (CNS). Emergency treatment consists of administration of pyridiniumoxime antidotes for reactivation of AChE. Yet, none of them are effective reactivators of all OP-inhibited AChE and their efficiency against phosphoramidate (tabun) is limited. Moreover, these oximes do not readily cross the Blood-Brain-Barrier (BBB) to reactivate AChE in the central nervous system. Furthermore, none of them are efficient on OP-inhibited butyrylcholinesterase (BChE), the back-up enzyme to AChE. As a consequence, remediation of both acute and chronic intoxications of civilian and military populations by OP continues to be a challenge of paramount importance. Recent crystallographic studies provided insight into the interactions between reactivator molecules within the AChE active site, revealing that oxime antidotes possessing vastly improved efficiency could be discovered through rational design. Around the world, teams are engaged in the design of new reactivators of OP-inhibited AChE. Teams from the EU, gathered in this consortium, have made the most promising discoveries. Our goal is thus to develop more efficient antidotes and improve the medical treatment of poisoning by highly toxic OP. The objective of this innovative project is to discover new multifunctional reactivators of AChE- and BuChE inhibited by organophosphorus nerve agents. In order to successfully achieve the objective of this exciting and challenging European project, the skills of scientists with skills spanning several disciplines will be required. Critically, the consortium also has access to facilities able to work with OP nerve agents safely.