Final Report Summary - FIT (Advanced web tool for Isothermel Titration Calorimetry data analysis and managent)
Microtubules are highly dynamic polymers of tubulin which play a key role in numerous vital cell processes including cell division, migration and axons stability. This dynamics, which is tightly regulated by microtubule associated proteins (MAPs) such as tau and stathmin, is also the target of a whole class of anticancer drugs known as microtubule targeting agents (MTAs). Because of the central role of microtubule associated proteins (MAPs) in many physiological processes and their interplay with anticancer drugs, they have been extensively studied. Nevertheless, the mechanisms of their interaction and sometimes even their binding to its primary target – tubulin - are still unclear. To solve these problems in the frame of FIT project we aim to achieve the following specific objectives: develop web-based software for advanced analysis of ITC data; decipher the mechanisms of tau binding to microtubules and the impact of MTAs on this interaction; study the impact of oxidative stress and zinc ions on tau binding to microtubules. Thus, the project consists of two parts experimental and software development.
Tau, a naturally unfolded protein that binds to tubulin by several highly homologous regions, promotes tubulin assembly into microtubules and stabilizes them. These homologous regions called repeats or microtubule-binding regions (MTBRs), are connected by flexible linkers and thus could bind to regular microtubule lattice with significantly different topology. Interaction of tau fragments with free tubulin and different forms of tubulin polymers, including microtubules and protofilaments was studied using isothermal titration calorimetry (ITC), differential scanning fluorimetry (nanoDSF) and turbodimetry. Totally it was expressed and purified ten different tau fragments, which consist of different number of MTBR regions. The binding of free MTBR regions to tubulin has very low enthalpy so could not be measured. Double and triple MTBR containing fragments also binds to tubulin with low but stull detectable enthalpy. It allowed us to determine that only fragment that consist of MTBR2 and MTBR3 binds to tubulin with formation one to one complex. Titration of free tubulin by other MTBR containing fragments results in biphasic binding curve. Basing on obtained results we propose new tau-tubulin binding model. According to this model unstructured tau molecule folds in half between MTBR2 and MTBR3 upon interaction with tubulin dimers in such way that MTBR1 and MTBR2 becomes anti-parallel to MTBR4 and MTBR3 respectively. Thus, MTBR2 and MTBR3 interacts with one tubulin dimes, and MTBR1 and MTBR4 with another. The stoichiometry of such complex tau:tubulin is equal to 1:2.
Our findings about the mode of tau binding to microtubule lattice fit well with recently published NMR study of tau-tubulin complex. This complex resemble the one of tubulin with stathmin wherein the last one also binds two tubulin dimers. Still stathmin is known as protein which induce microtubules depolymerization, while tau in contrast favor tubulin polymerization. The key for understanding why the formation of two similar complexes leads to opposite effects could be found in the structure of these complexes, which is in turn could be explained by the properties of stathmin and tau per se. Indeed, in both complexes two tubulin dimers bind longitudinally but since stathmin represents a long rigid and curved α-helix, tubulin dimers are oriented at a fixed angle to each other, which do not allow them to be integrated in microtubules’ regular structure. Otherwise unstructured flexible tau allow tubulin dimers to do it. Our math simulation shows that just the the formation of tightly bound two dimers of tubulin lead to the shift of equilibrium towards microtubules formation. Such effect could be explained by an increase of the constant of association of tau-tubulin complex in comparison with free tubulin dimer to neighbor protofilaments. Thus our data not only allowed us to propose the model of tau-tubulin interaction, but also to explain the common molecular mechanism which lays in the regulation of tubulin polymerization by MAPs.
Web tool for ITC data analysis has been developed using DHTMLX JavaScript widgets library. Web tool allow to upload raw ITC data files on server and manage them. It also allows to do preliminary raw data processing such as instrumental baseline creation, manipulation and so on. Tool allow to preview uploaded raw data in different forms and associate with them experimental conditions, which permits further search experimental curves by different parameters. It is possible to fit uploaded experimental data with several standard binding models. Still during assembling and testing web application on the project server revealed a numerous bug, which will be fixed during next year.
http://fit.microcalorimetry-marseille.com/
Tau, a naturally unfolded protein that binds to tubulin by several highly homologous regions, promotes tubulin assembly into microtubules and stabilizes them. These homologous regions called repeats or microtubule-binding regions (MTBRs), are connected by flexible linkers and thus could bind to regular microtubule lattice with significantly different topology. Interaction of tau fragments with free tubulin and different forms of tubulin polymers, including microtubules and protofilaments was studied using isothermal titration calorimetry (ITC), differential scanning fluorimetry (nanoDSF) and turbodimetry. Totally it was expressed and purified ten different tau fragments, which consist of different number of MTBR regions. The binding of free MTBR regions to tubulin has very low enthalpy so could not be measured. Double and triple MTBR containing fragments also binds to tubulin with low but stull detectable enthalpy. It allowed us to determine that only fragment that consist of MTBR2 and MTBR3 binds to tubulin with formation one to one complex. Titration of free tubulin by other MTBR containing fragments results in biphasic binding curve. Basing on obtained results we propose new tau-tubulin binding model. According to this model unstructured tau molecule folds in half between MTBR2 and MTBR3 upon interaction with tubulin dimers in such way that MTBR1 and MTBR2 becomes anti-parallel to MTBR4 and MTBR3 respectively. Thus, MTBR2 and MTBR3 interacts with one tubulin dimes, and MTBR1 and MTBR4 with another. The stoichiometry of such complex tau:tubulin is equal to 1:2.
Our findings about the mode of tau binding to microtubule lattice fit well with recently published NMR study of tau-tubulin complex. This complex resemble the one of tubulin with stathmin wherein the last one also binds two tubulin dimers. Still stathmin is known as protein which induce microtubules depolymerization, while tau in contrast favor tubulin polymerization. The key for understanding why the formation of two similar complexes leads to opposite effects could be found in the structure of these complexes, which is in turn could be explained by the properties of stathmin and tau per se. Indeed, in both complexes two tubulin dimers bind longitudinally but since stathmin represents a long rigid and curved α-helix, tubulin dimers are oriented at a fixed angle to each other, which do not allow them to be integrated in microtubules’ regular structure. Otherwise unstructured flexible tau allow tubulin dimers to do it. Our math simulation shows that just the the formation of tightly bound two dimers of tubulin lead to the shift of equilibrium towards microtubules formation. Such effect could be explained by an increase of the constant of association of tau-tubulin complex in comparison with free tubulin dimer to neighbor protofilaments. Thus our data not only allowed us to propose the model of tau-tubulin interaction, but also to explain the common molecular mechanism which lays in the regulation of tubulin polymerization by MAPs.
Web tool for ITC data analysis has been developed using DHTMLX JavaScript widgets library. Web tool allow to upload raw ITC data files on server and manage them. It also allows to do preliminary raw data processing such as instrumental baseline creation, manipulation and so on. Tool allow to preview uploaded raw data in different forms and associate with them experimental conditions, which permits further search experimental curves by different parameters. It is possible to fit uploaded experimental data with several standard binding models. Still during assembling and testing web application on the project server revealed a numerous bug, which will be fixed during next year.
http://fit.microcalorimetry-marseille.com/