Ancestral Allostery – AlloAnc

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.