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The discovery that a large part of the proteomes has a high-degree of structural disorder has raised new challenges in understanding how proteins lacking of stable secondary structure perform their function. Indeed, the so-called intrinsically disordered proteins (IDPs) violate the paradigm by which function necessarily relates to structure. The ability of computer simulations to sample dynamics of molecules at the nanometer resolution, can be extremely useful for our understanding of IDPs’ function and dynamics. However, molecular dynamics simulations, that for decades have relied on force fields tuned to reproduce the correct behavior of structured proteins, fail for intrinsically disordered proteins by yielding ensembles that are too collapsed.

Some attempts pointing towards the rescaling of protein-water interactions and the re-parameterization of water have been made with promising outcomes. Nevertheless, a completely re-parameterized force field able to reproduce the correct dynamics of structured and intrinsically disordered proteins would be ideal.

I have shown that a new force field parametrised on the basis of the Kirkwood-Buff theory of solutions is able to correctly reproduce the dimensions of the ensemble of a particularly collapse-prone fragment of Nucleoporin 153. In combination with smFRET and SAXS experiments, we were able to show that following the Kirkwood-Buff theory is a valid method to solve the problem of over collapse and current efforts are under way to improve the KBFF and finally develop a force field able to simulate structured and unstructured molecules.

Read more about it in our paper that you can find on the page listing my publications!

Another useful link is:

The Kirkwood-Buff force field page.


The ability of intrinsically disordered proteins to perform complex functions is most often related to their capacity to bind structured targets inside cells. Two main mechanisms of binding have been identified so far: conformational selection and induced fit. In a conformational selection scenario across the large set of conformations that IDPs explore, some (which are particularly prone to bind the target) are more explored, maximising the probability of correctly engaging the binding partner. On the other hand, the induced fit mechanism prefigures the appearance of some structured elements in the IDP upon binding the partner. In both cases, some, even minimal formation of secondary structure elements is required for the binding.

Schematic representation of Importinβ molecules approaching the nuclear pore complex
Schematic representation of Importinβ molecules approaching the nuclear pore complex

Some intrinsically disordered proteins have nevertheless, the ability to perform their function in complete absence of secondary structure. It is the case of nucleoporins that form a mesh in nuclear pore complexes and allow the selective passage of molecular components in & out of the nucleus, by binding specific nuclear transport receptors through FG motifs that dock into the hydrophobic pockets on the receptor’s surface. Intriguingly, the trespassing of the NTRs through the nuclear pore is extremely fast (in the ms timescale) considering the amount of interactions that NTRs and nucleoporins must engage. This particularity creates the so called transport paradox: how thousands of interactions can be specifically made and broken in such a fast timescale?

 

 

Summary of the interdisciplinary approaches used to discover and quantify the binding mechanisms of Importinβ to Nup153
Summary of the interdisciplinary approaches used to discover and quantify the binding mechanisms of Importinβ to Nup153

We have found the mechanisms regulating such a paradox by using a highly interdisciplinary computational and experimental approach for the study of intrinsically disordered nucleoporins and NTRs. Our results opened to the understanding of a new mechanism by which efficient and fast binding are not regulated by neither a conformational selection nor induced fit mechanism but by the presence of numerous, minimalistic binding moieties (the FG-repeats) along the nucleoporin’s sequence.

Molecular Dynamics simulations revealed that the association between binding moieties is among the fastest ever recorded and that the affinity of the single interactions are very weak. Through weak interactions fast association and dissociation rates can explain the fast transport that physiological regulates the transportation of molecular components across cellular compartments.


  • Mechanical activation of kinases
closed Src Kinase in complex with p130Cas peptide
closed Src Kinase in complex with p130Cas peptide

Kinases belong one of the most interesting class of proteins as they regulate a myriad of cellular processes. Recently, kinases have also been identified in cellular processes that require the application and propagation of force and such observations have sparked the idea that some kinases may actually act as mechano-sensors inside the cell and transduce the application of force into cellular signals useful to maintain the correct physiology of the cell.

Although the role of some kinases as mechanosensors is clear, the mechanism through which the force-driven activation of kinases is achieved is still unknown. Recently, the Molecular Biomechanics Group at HITS has revealed the basis regulating the force-driven activation of the focal adhesion kinase (FAK). You can read the published work about this here. There are other kinases with important roles in cell’s physiology that may be activated and regulated by force and I am currently performing computer simulations in order to reveal the details and the mechanisms of force-driven activation of some of these molecules.


Pectin methyl esterase enzymes (PMEs) catalyse the removal of methyl groups from long, linear pectin chains composing the plant cell wall. This modification is essential for the development and survival of plants as the methyl groups removal promotes the formation of negatively charged polymers useful for remodelling the plant cell wall during the developmental stages of the plants’ life. Bacterial and fungi also express PMEs as the modification of pectin leads the way to pectinolytic enzymes aiming to the destruction of the plant cell wall.

Pectin methyl esterase in complex to a 10-mer galacturonan oligomer. The protein's surface is coloured according to its electrostatic potential.
Pectin methyl esterase in complex to a 10-mer galacturonan oligomer. The protein’s surface is coloured according to its electrostatic potential. The zoom-in represents the active site of the enzyme with flanking subsites highlighted.

Looking at their structure, PMEs are carbohydrate-binding enzymes that feature the presence of a binding groove able to allocate polysaccharides. The binding groove can be subdivided in smaller subsites, each one docking monosaccharide units of the polymer. While a single subsite contains the catalytic triad able to catalyse the de-methylesterification, other subsites are place holders for the remaining monosaccharides.

Interestingly, different PMEs have shown the ability to produce continuously de-methylated stretches along the pectin polymers suggesting a processive action through which the enzyme would perform several catalyses before dissociating from the substrate. More interestingly, while the majority of processive enzymes uses external energetic co-factors (such as ATP or GTP), PMEs are able to achieve processivity without external energy sources.

Understanding the mechanism through which PMEs perform processive catalysis without is therefore of great importance for controlling plant pathogenic infection and, at the same time, control PME activity in industrial processes where these enzymes found application.

Potential energy profile of a single de-methylesterification cycle of a processive pectin methyl esterase
Potential energy profile of a single de-methylesterification cycle of a processive pectin methyl esterase

In some of my published work, we have found the basis of PMEs processivity by characterising the motions of the freshly de-methylesterified product of reaction into the binding groove. The internal motions of the product favour the repositioning of the monosaccharides for further de-methylesterification events. You can read more about this story here!

Breathing motions of the enzyme are, on the other hand, responsible for the sliding of the protein along the polymeric chain. You can read more about the role of electrostatic and breathing motion of PMEs here!

 

This project is in collaboration with The University of Auckland and Massey University in New Zealand.


  • Pectin methyl esterase inhibitors: test cases for studying micro environmentally tuned protein-protein interaction.  

Pectin methyl esterase inhibitors (PMEIs) are a set of proteins largely expressed in higher plants and control the activity of Pectin Methylesterase enzymes (PMEs).

Complex between PME3 (green) and PMEI7 (blue and red at pH 5 and 7 respectively). The represented conformations have been sampled using molecular dynamics simulations of the modelled association. At pH 7 the association is weaker.
Complex between PME3 (green) and PMEI7 (blue and red at pH 5 and 7 respectively). The represented conformations have been sampled using molecular dynamics simulations of the modelled association. At pH 7 the association is weaker.

Although their importance in the modulation of plant metabolism is clear, the reason for which tens of different isoforms are selectively expressed in different plant tissues is still unknown. In A. thaliana, for example, 66 different PMEI isoforms have been identified and most interestingly they seem to have a different functional efficiency in different micro-environmental conditions such as different pH or ionic strength.

This makes PMEIs highly interesting to understand how protein-protein binding can be finely tuned with respect to different micro-environmental conditions.

Above all, is it possible to understand the molecular basis characterising the interaction of independent and pH/ionic strength independent PME-PMEIs interactions? Is it possible to rationally modulate PME-PMEIs interactions to control the inhibition in vitro, vivo and in industrial processes such as, for example, juice clarification?.

This ability would have a great impact on both basic and applied research.

Molecular dynamics simulations performed on recently characterized PMEI isoforms from A. thaliana are being employed to investigate the PMEI-PME complex formation in different micro environmental conditions. This project is in collaboration with Prof. Jerome Pelloux at the University of Picardie.