Francesco Luigi Gervasio

Metadynamics: a method to simulate rare events and reconstruct the free energy in biophysics, chemistry and material science

Metadynamics is a powerful algorithm that can be used both for reconstructing the free energy and for accelerating rare events in systems described by complex Hamiltonians, at the classical or at the quantum level. In the algorithm the normal evolution of the system is biased by a history-dependent potential constructed as a sum of Gaussians centered along the trajectory followed by a suitably chosen set of collective variables. The sum of Gaussians is exploited for reconstructing iteratively an estimator of the free energy and forcing the system to escape from local minima. This review is intended to provide a comprehensive description of the algorithm, with a focus on the practical aspects that need to be addressed when one attempts to apply metadynamics to a new system: (i) the choice of the appropriate set of collective variables; (ii) the optimal choice of the metadynamics parameters and (iii) how to control the error and ensure convergence of the algorithm.

From A to B in free energy space

The authors present a new method for searching low free energy paths in complex molecular systems at finite temperature. They introduce two variables that are able to describe the position of a point in configurational space relative to a preassigned path. With the help of these two variables the authors combine features of approaches such as metadynamics or umbrella sampling with those of path based methods. This allows global searches in the space of paths to be performed and a new variational principle for the determination of low free energy paths to be established. Contrary to metadynamics or umbrella sampling the path can be described by an arbitrary large number of variables, still the energy profile along the path can be calculated. The authors exemplify the method numerically by studying the conformational changes of alanine dipeptide.

Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity.

Effects of oncogenic mutations on the conformational free-energy landscape of EGFR kinase

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase are frequently found in many cancers. It has been suggested that changes in the equilibrium between its active and inactive conformations are linked to its oncogenic potential. Here, we quantify the effects of some of the most common single (L858R and T790M) and double (T790M-L858R) oncogenic mutations on the conformational free-energy landscape of the EGFR kinase domain by using massive molecular dynamics simulations together with parallel tempering, metadynamics, and one of the best force-fields available. Whereas the wild-type EGFR catalytic domain monomer is mostly found in an inactive conformation, our results show a clear shift toward the active conformation for all of the mutants. The L858R mutation stabilizes the active conformation at the expense of the inactive conformation and rigidifies the αC-helix. The T790M gatekeeper mutant favors activation by stabilizing a hydrophobic cluster. Finally, T790M with L858R shows a significant positive epistasis effect. This combination not only stabilizes the active conformation, but in nontrivial ways changes the free-energy landscape lowering the transition barriers.

Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

The Unfolded Ensemble and Folding Mechanism of the C-Terminal GB1 β-Hairpin

In this work, we shed new light on a much-studied case of beta-hairpin folding by means of advanced molecular dynamics simulations. A fully atomistic description of the protein and the solvent molecule is used, together with metadynamics, to accelerate the sampling and estimate free-energy landscapes. This is achieved using the path collective variables approach, which provides an adaptive description of the mechanism under study. We discover that the folding mechanism is a multiscale process where the turn region conformation leads to two different energy pathways that are connected by elongated structures. The former displays a stable 2:4 native-like structure in which an optimal hydrophobic packing and hydrogen bond pattern leads to 8 kcal/mol of stabilization. The latter shows a less-structured 3:5 beta-sheet, where hydrogen bonds and hydrophobic packing provide only 2.5 kcal/mol of stability. This perspective is fully consistent with experimental evidence that shows this to be a prototypical two-state folder, while it redefines the nature of the unfolded state.

New advances in metadynamics

Metadynamics is an algorithm for accelerating rare events and reconstructing the associated free energy surface. It works by biasing the evolution of the system by a history‐dependent potential that is adaptively constructed in the space of a suitably chosen set of collective variables. Since its first appearance, the method has been successfully applied in several domains of science. Its widespread adoption is not only due to its efficiency, flexibility, and availability but also to its continuous evolution and its combination with complementary enhanced sampling algorithms. Here, we focus on the progress made in the development of more general and powerful collective variables and on the very recent and exciting evolutions of the method.

The different flexibility of c-Src and c-Abl kinases regulates the accessibility of a druggable inactive conformation.

c-Src and c-Abl are two closely related protein kinases that constitute important anticancer targets. Despite their high sequence identity, they show different sensitivities to the anticancer drug imatinib, which binds specifically to a particular inactive conformation in which the Asp of the conserved DFG motif points outward (DFG-out). We have analyzed the DFG conformational transition of the two kinases using massive molecular dynamics simulations, free energy calculations, and isothermal titration calorimetry. On the basis of the reconstruction of the free energy surfaces for the DFG-in to DFG-out conformational changes of c-Src and c-Abl, we propose that the different flexibility of the two kinases results in a different stability of the DFG-out conformation and might be the main determinant of imatinib selectivity.

Molecular Mechanism of SSR128129E, an Extracellularly Acting, Small-Molecule, Allosteric Inhibitor of FGF Receptor Signaling

The fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling network plays an important role in cell growth, survival, differentiation, and angiogenesis. Deregulation of FGFR signaling can lead to cancer development. Here, we report an FGFR inhibitor, SSR128129E (SSR), that binds to the extracellular part of the receptor. SSR does not compete with FGF for binding to FGFR but inhibits FGF-induced signaling linked to FGFR internalization in an allosteric manner, as shown by crystallography studies, nuclear magnetic resonance, Fourier transform infrared spectroscopy, molecular dynamics simulations, free energy calculations, structure-activity relationship analysis, and FGFR mutagenesis. Overall, SSR is a small molecule allosteric inhibitor of FGF/FGFR signaling, acting via binding to the extracellular part of the FGFR.

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein-DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo.