Deciphering the molecular basis of VDAC-Hexokinase interaction

Hexokinase (HK) is the first enzyme of glycolytic pathway and traps glucose inside the cell as 6Pi-glucose. To conveniently access ATP necessary for phosphorylation, HK binds on the surface of mitochondria, using Voltage Dependent Anion selective Channel (VDAC) both as a docking site and as the pore through which ATP is exported outside the mitochondria. The binding HK-VDAC has been identified as an important determinant in the apoptotic balance of the mitochondrial pathway and, consequently, as a cancerous cell hallmark. This situation was described as Warburg effect, i.e. the prevalence of anaerobic glycolysis, fueled by HK, against the aerobic glycolysis that utilizes the mitochondrial enzymatic activities. It has even become the tool to map the presence of active cancer in PET diagnostic, where a radioactive analogue of HK substrate is followed to associate cancer metabolism to cell districts. Unfortunately, the mechanism of binding between HK and VDAC is still unknown. HK is a soluble enzyme provided with a short N-terminal hydrophobic tail (the N-anchor) whose deletion hinders the binding of HK to the organelle. Deletion of VDAC E73, an acid residue exposed to the hydrophobic section of membrane bilayer, abolishes the binding between the two proteins. Most recently, molecular simulations questioned such result and proposed a new model of HK-VDAC binding. In this project we will definitely localize the residues allowing the binding between VDAC1 and HKI. The identification of the residues involved in the binding will be performed with two similar but different strategies: i) by producing VDAC1 mutants with cysteine residues located in putative binding areas and, on the opposite, by including cysteines in synthetic peptides with the N-anchor sequence (NHK) and purposing the two actors to react by oxidation and disulfide formation; ii) in another strategy, the whole HKI will be used and the binding will be produced by exploiting chemical cross-linking reagents or by reactions with click chemistry.

These two approaches will heavily relay on bioinformatics and molecular modelling work. The in-silico analysis will apply docking and more sophisticated molecular tools to examine not only the interaction between the N-anchor and the pore wall, but also those between the hydrophilic surfaces of HK and, as a counterpart, the exposed cytosolic loops of VDAC. It will be used both to design experiments and to interpret their outputs. The final validation of the obtained results will be produced by introducing modified proteins in cellulo and analyzing their influence on life vitality and mitochondrial polarization.
The achievement of the goal, even though only partial, with the identification of a few residues confidentially involved in the interaction, would be a very important success since VDAC-HK interaction is a main target of proposed therapeutic strategies in relevant pathologies like cancer and neurodegenerative diseases.