Pliability in the m6A-Binding Region Extends Druggability of YTH Domains.

Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m6A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.

Probing the conformational dynamics of an Ago-RNA complex in water/methanol solution.

Argonaute (Ago) proteins mediate target recognition guiding miRNA to bind complementary mRNA primarily in the seed region. However, additional pairing can occur beyond the seed, forming a supplementary duplex that can contribute to the guide-target affinity. In order to shed light on the connection, between protein-RNA interactions and miRNA-mRNA seed and supplementary duplex mobility, we carried out molecular dynamics simulations at the microsecond time-scale using a different approach compared to the ones normally used. Until now, theoretical investigations with classical MD on Ago-RNA complexes have been focused primarily on pure water solvent, which mimics the natural environment of biological molecules. Here, we explored the conformational space of a human Ago2 (hAgo2) bound to the seed + supplementary miRNA-mRNA duplex, using the solvent environment as a molecular probe. MD simulations have been performed in a mixture of water/MeOH at a molar ratio of 70 : 30 as well as in pure water for comparison. Our findings revealed that the mixed solvent promotes protein RNA association, principally enhancing salt-linkages between basic amino acid side-chains and acidic phosphates of the sugar-phosphate backbone. The primary effect registered was the restriction of supplementary duplex flexibility and the stabilization of the miRNA 3' terminus. Interestingly, we observed that the influence of the solvent appears to have almost no impact on the conformation of the seed duplex.

Conformational response to ligand binding of TMPRSS2, a protease involved in SARS-CoV-2 infection: Insights through computational modeling.

Thanks to the considerable research which has been undertaken in the last few years to improve our understanding of the biology and mechanism of action of SARS-CoV-2, we know how the virus uses its surface spike protein to infect host cells. The transmembrane prosthesis, serine 2 (TMPRSS2) protein, located on the surface of human cells, recognizes the cleavage site in the spike protein, leading to the release of the fusion peptide and entry of the virus into the host cells. Because of its role, TMPRSS2 has been proposed as a drug target to prevent infection by the virus. In this study, we aim to increase our understanding of TMPRSS2 using long scale microsecond atomistic molecular dynamics simulations, focusing on the conformational changes over time. The comparison between simulations conducted on the protein in the native (apo) and inhibited form (holo), has shown that in the holo form the inhibitor stabilizes the catalytic site and induces rearrangements in the extracellular domain of the protein. In turn, it leads to the formation of a new cavity in the vicinity of the ligand binding pocket that is stable in the microsecond time scale. Given the low specificity of known protease inhibitors, these findings suggest a new potential drug target site that can be used to improve TMPRSS2 specific recognition by newly designed inhibitors.

Isoamphipathic antibacterial molecules regulating activity and toxicity through positional isomerism.

Peptidomimetic antimicrobials exhibit a selective interaction with bacterial cells over mammalian cells once they have achieved an optimum amphiphilic balance (hydrophobicity/hydrophilicity) in the molecular architecture. To date, hydrophobicity and cationic charge have been considered the crucial parameters to attain such amphiphilic balance. However, optimization of these properties is not enough to circumvent unwanted toxicity towards mammalian cells. Hence, herein, we report new isoamphipathic antibacterial molecules (IAMs: 1-3) where positional isomerism was introduced as one of the guiding factors for molecular design. This class of molecules displayed good (MIC = 1-8 µg mL-1 or µM) to moderate [MIC = 32-64 µg mL-1 (32.2-64.4 µM)] antibacterial activity against multiple Gram-positive and Gram-negative bacteria. Positional isomerism showed a strong influence on regulating antibacterial activity and toxicity for ortho [IAM-1: MIC = 1-32 µg mL-1 (1-32.2 µM), HC50 = 650 µg mL-1 (654.6 µM)], meta [IAM-2: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 98 µg mL-1 (98.7 µM)] and para [IAM-3: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 160 µg mL-1 (161.1 µM)] isomers. Co-culture studies and investigation of membrane dynamics indicated that ortho isomer, IAM-1 exerted more selective activity towards bacterial over mammalian membranes, compared to meta and para isomers. Furthermore, the mechanism of action of the lead molecule (IAM-1) has been characterized through detailed molecular dynamics simulations. In addition, the lead molecule displayed substantial efficacy against dormant bacteria and mature biofilms, unlike conventional antibiotics. Importantly, IAM-1 exhibited moderate in vivo activity against MRSA wound infection in a murine model with no detectable dermal toxicity. Altogether, the report explored the design and development of isoamphipathic antibacterial molecules to establish the role of positional isomerism in achieving selective and potential antibacterial agents.

Computational Study of Helicase from SARS-CoV-2 in RNA-Free and Engaged Form.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic that broke out in 2020 and continues to be the cause of massive global upheaval. Coronaviruses are positive-strand RNA viruses with a genome of ~30 kb. The genome is replicated and transcribed by RNA-dependent RNA polymerase together with accessory factors. One of the latter is the protein helicase (NSP13), which is essential for viral replication. The recently solved helicase structure revealed a tertiary structure composed of five domains. Here, we investigated NSP13 from a structural point of view, comparing its RNA-free form with the RNA-engaged form by using atomistic molecular dynamics (MD) simulations at the microsecond timescale. Structural analyses revealed conformational changes that provide insights into the contribution of the different domains, identifying the residues responsible for domain-domain interactions in both observed forms. The RNA-free system appears to be more flexible than the RNA-engaged form. This result underlies the stabilizing role of the nucleic acid and the functional core role of these domains.

Role of the membrane anchor in the regulation of Lck activity.

Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 transmembrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.

Altered Local Interactions and Long-Range Communications in UK Variant (B.1.1.7) Spike Glycoprotein.

The COVID-19 pandemic is caused by SARS-CoV-2. Currently, most of the research efforts towards the development of vaccines and antibodies against SARS-CoV-2 were mainly focused on the spike (S) protein, which mediates virus entry into the host cell by binding to ACE2. As the virus SARS-CoV-2 continues to spread globally, variants have emerged, characterized by multiple mutations of the S glycoprotein. Herein, we employed microsecond-long molecular dynamics simulations to study the impact of the mutations of the S glycoprotein in SARS-CoV-2 Variant of Concern 202012/01 (B.1.1.7), termed the "UK variant", in comparison with the wild type, with the aim to decipher the structural basis of the reported increased infectivity and virulence. The simulations provided insights on the different dynamics of UK and wild-type S glycoprotein, regarding in particular the Receptor Binding Domain (RBD). In addition, we investigated the role of glycans in modulating the conformational transitions of the RBD. The overall results showed that the UK mutant experiences higher flexibility in the RBD with respect to wild type; this behavior might be correlated with the increased transmission reported for this variant. Our work also adds useful structural information on antigenic "hotspots" and epitopes targeted by neutralizing antibodies.

Phosphorylation, Mg-ADP, and Inhibitors Differentially Shape the Conformational Dynamics of the A-Loop of Aurora-A.

The conformational state of the activation loop (A-loop) is pivotal for the activity of most protein kinases. Hence, the characterization of the conformational dynamics of the A-loop is important to increase our understanding of the molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. Here, we carry out a combination of molecular dynamics (MD) and essential dynamics (ED) analyses to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the A-loop of Aurora-A. MD revealed that the stability of the A-loop in an open conformation is enhanced by single phospho-Thr-288, while paradoxically, the presence of a second phosphorylation at Thr-287 decreases such stability and renders the A-loop more fluctuant in time and space. Moreover, we found that this post-translational modification has a significant effect on the direction of the A-loop motions. ED analysis suggests that the presence of the phosphate moiety induces the dynamics of Aurora-A to sample two distinct energy minima, instead of a single large minimum, as in unphosphorylated Aurora-A states. This observation indicates that the conformational distributions of Aurora-A with both single and double phospho-threonine modifications are remarkably different from the unphosphorylated state. In the closed states, binding of CD532 and MLN8054 inhibitors has the effect of increasing the distance of the N- and C-lobes of the kinase domain of Aurora-A, and the angle analysis between those two lobes during MD simulations showed that the N- and C-lobes are kept more open in presence of CD532, compared to MLN8054. As the A-loop is a common feature of Aurora protein kinases, our studies provide a general description of the conformational dynamics of this structure upon phosphorylation and different ligands binding.

Computational Studies of SARS-CoV-2 3CLpro: Insights from MD Simulations.

Given the enormous social and health impact of the pandemic triggered by severe acute respiratory syndrome 2 (SARS-CoV-2), the scientific community made a huge effort to provide an immediate response to the challenges posed by Coronavirus disease 2019 (COVID-19). One of the most important proteins of the virus is an enzyme, called 3CLpro or main protease, already identified as an important pharmacological target also in SARS and Middle East respiratory syndrome virus (MERS) viruses. This protein triggers the production of a whole series of enzymes necessary for the virus to carry out its replicating and infectious activities. Therefore, it is crucial to gain a deeper understanding of 3CLpro structure and function in order to effectively target this enzyme. All-atoms molecular dynamics (MD) simulations were performed to examine the different conformational behaviors of the monomeric and dimeric form of SARS-CoV-2 3CLpro apo structure, as revealed by microsecond time scale MD simulations. Our results also shed light on the conformational dynamics of the loop regions at the entry of the catalytic site. Studying, at atomic level, the characteristics of the active site and obtaining information on how the protein can interact with its substrates will allow the design of molecules able to block the enzymatic function crucial for the virus.

Insights into PPARγ Phosphorylation and Its Inhibition Mechanism.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.

Revisiting the "satisfaction of spatial restraints" approach of MODELLER for protein homology modeling.

The most frequently used approach for protein structure prediction is currently homology modeling. The 3D model building phase of this methodology is critical for obtaining an accurate and biologically useful prediction. The most widely employed tool to perform this task is MODELLER. This program implements the "modeling by satisfaction of spatial restraints" strategy and its core algorithm has not been altered significantly since the early 1990s. In this work, we have explored the idea of modifying MODELLER with two effective, yet computationally light strategies to improve its 3D modeling performance. Firstly, we have investigated how the level of accuracy in the estimation of structural variability between a target protein and its templates in the form of σ values profoundly influences 3D modeling. We show that the σ values produced by MODELLER are on average weakly correlated to the true level of structural divergence between target-template pairs and that increasing this correlation greatly improves the program's predictions, especially in multiple-template modeling. Secondly, we have inquired into how the incorporation of statistical potential terms (such as the DOPE potential) in the MODELLER's objective function impacts positively 3D modeling quality by providing a small but consistent improvement in metrics such as GDT-HA and lDDT and a large increase in stereochemical quality. Python modules to harness this second strategy are freely available at https://github.com/pymodproject/altmod. In summary, we show that there is a large room for improving MODELLER in terms of 3D modeling quality and we propose strategies that could be pursued in order to further increase its performance.

Cysteine 180 Is a Redox Sensor Modulating the Activity of Human Pyridoxal 5'-Phosphate Histidine Decarboxylase.

Histidine decarboxylase is a pyridoxal 5'-phosphate enzyme catalyzing the conversion of histidine to histamine, a bioactive molecule exerting its role in many modulatory processes. The human enzyme is involved in many physiological functions, such as neurotransmission, gastrointestinal track function, cell growth, and differentiation. Here, we studied the functional properties of the human enzyme and, in particular, the effects exerted at the protein level by two cysteine residues: Cys-180 and Cys-418. Surprisingly, the enzyme exists in an equilibrium between a reduced and an oxidized form whose extent depends on the redox state of Cys-180. Moreover, we determined that (i) the two enzymatic redox species exhibit modest structural changes in the coenzyme microenvironment and (ii) the oxidized form is slightly more active and stable than the reduced one. These data are consistent with the model proposed by bioinformatics analyses and molecular dynamics simulations in which the Cys-180 redox state could be responsible for a structural transition affecting the C-terminal domain reorientation leading to active site alterations. Furthermore, the biochemical properties of the purified C180S and C418S variants reveal that C180S behaves like the reduced form of the wild-type enzyme, while C418S is sensitive to reductants like the wild-type enzyme, thus allowing the identification of Cys-180 as the redox sensitive switch. On the other hand, Cys-418 appears to be a residue involved in aggregation propensity. A possible role for Cys-180 as a regulatory switch in response to different cellular redox conditions could be suggested.

Molecular dynamics recipes for genome research.

Molecular dynamics (MD) simulation allows one to predict the time evolution of a system of interacting particles. It is widely used in physics, chemistry and biology to address specific questions about the structural properties and dynamical mechanisms of model systems. MD earned a great success in genome research, as it proved to be beneficial in sorting pathogenic from neutral genomic mutations. Considering their computational requirements, simulations are commonly performed on HPC computing devices, which are generally expensive and hard to administer. However, variables like the software tool used for modeling and simulation or the size of the molecule under investigation might make one hardware type or configuration more advantageous than another or even make the commodity hardware definitely suitable for MD studies. This work aims to shed lights on this aspect.

Lethal digenic mutations in the K+ channels Kir4.1 (KCNJ10) and SLACK (KCNT1) associated with severe-disabling seizures and neurodevelopmental delay.

A 2-yr-old boy presented profound developmental delay, failure to thrive, ataxia, hypotonia, and tonic-clonic seizures that caused the death of the patient. Targeted and whole exome sequencing revealed two heterozygous missense variants: a novel mutation in the KCNJ10 gene that encodes for the inward-rectifying K+ channel Kir4.1 and another previously characterized mutation in KCNT1 that encodes for the Na+-activated K+ channel known as Slo2.2 or SLACK. The objectives of this study were to perform the clinical and genetic characterization of the proband and his family and to examine the functional consequence of the Kir4.1 mutation. The mutant and wild-type KCNJ10 constructs were generated and heterologously expressed in Xenopus laevis oocytes, and whole cell K+ currents were measured using the two-electrode voltage-clamp technique. The KCNJ10 mutation c.652C>T resulted in a p.L218F substitution at a highly conserved residue site. Wild-type KCNJ10 expression yielded robust Kir current, whereas currents from oocytes expressing the mutation were reduced, remarkably. Western Blot analysis revealed reduced protein expression by the mutation. Kir5.1 subunits display selective heteromultimerization with Kir4.1 constituting channels with unique kinetics. The effect of the mutation on Kir4.1/5.1 channel activity was twofold: a reduction in current amplitudes and an increase in the pH-dependent inhibition. We thus report a novel loss-of-function mutation in Kir4.1 found in a patient with a coexisting mutation in SLACK channels that results in a fatal disease.NEW & NOTEWORTHY We present and characterize a novel mutation in KCNJ10 Unlike previously reported EAST/SeSAME patients, our patient was heterozygous, and contrary to previous studies, mimicking the heterozygous state by coexpression resulted in loss of channel function. We report in the same patient co-occurrence of a KCNT1 mutation resulting in a more severe phenotype. This study provides new insights into the phenotypic spectrum and to the genotype-phenotype correlations associated with EAST/SeSAME and MMFSI.

Thermal compaction of the intrinsically disordered protein tau: entropic, structural, and hydrophobic factors.

Globular denatured proteins have structural properties similar to those of random coils. Experiments on denatured proteins have shown that when the temperature is increased thermal compaction may take place, resulting in a reduction of their radius of gyration Rg to range between 5% and 35% of its initial value. This phenomenon has been attributed to various causes, namely entropic, hydrophobic, and structural factors. The intrinsically disordered protein tau, which helps in nucleating and stabilizing microtubules in the axons of the neurons, also undergoes a relevant compaction process: when its temperature is increased from 293 K to 333 K its gyration radius decreases by 18%. We have performed an atomistic simulation of this molecule, at the lowest and highest temperatures of the mentioned interval, using both standard molecular dynamics and metadynamics, in parallel with small-angle X-ray scattering experiments. Using the fit of the experimental data and a genetic algorithm to select the most probable configurations among those produced in both atomistic simulations (standard MD and metadynamics), we were able to compute relevant changes, related to the temperature increase, in the average angles between residues, in the transient secondary structures, in the solvent accessible surface area, and in the number of intramolecular H-bonds. The analysis of the data showed how to decompose the compaction phenomenon into three contributions. An estimate of the entropic contribution to the compaction was obtained using the changes in the mean values of the angles between contiguous residues. The computation of the solvent accessible surface at the two temperatures allowed an estimation of the second factor contributing to the compaction, namely the increase in the hydrophobic interaction. We also measured the change in the average number of residues temporarily being in α-helices, 3-helices, PP II helices, ß-sheets and ß-turns. Those changes in the secondary structure population produce a reduction in the contour length of the protein, yielding a structural contribution to the reduction of Rg. This analysis shows that in tau the entropic factor accounts for about 60% of the compaction, the hydrophobic factor for about 25%, and the change in the secondary structure for about 15%.

Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy.

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy.

Effect of DNA on the conformational dynamics of the endonucleases I-DmoI as provided by molecular dynamics simulations.

The conformational behavior of the wild-type endonucleases I-DmoI and two of its mutants has been studied in the presence and in the absence of DNA target sequences by means of extended molecular dynamics simulations. Our results show that in the absence of DNA, the three protein forms explore a similar essential conformational space, whereas when bound to the same DNA target sequence of 25 base pairs, they diversify and restrain the subspace explored. In addition, the differences in the essential subspaces explored by the residues near the catalytic site for both the bound and unbound forms are discussed in background of the experimental protein activity.

A Calsequestrin-1 Mutation Associated with a Skeletal Muscle Disease Alters Sarcoplasmic Ca2+ Release.

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy".

Voltage-sensor conformation shapes the intra-membrane drug binding site that determines gambierol affinity in Kv channels.

Marine ladder-shaped polyether toxins are implicated in neurological symptoms of fish-borne food poisonings. The toxin gambierol, produced by the marine dinoflagellate Gambierdiscus toxicus, belongs to the group of ladder-shaped polyether toxins and inhibits Kv3.1 channels with nanomolar affinity through a mechanism of gating modification. Binding determinants for gambierol localize at the lipid-exposed interface of the pore forming S5 and S6 segments, suggesting that gambierol binds outside of the permeation pathway. To explore a possible involvement of the voltage-sensing domain (VSD), we made different chimeric channels between Kv3.1 and Kv2.1, exchanging distinct parts of the gating machinery. Our results showed that neither the electro-mechanical coupling nor the S1-S3a region of the VSD affect gambierol sensitivity. In contrast, the S3b-S4 part of the VSD (paddle motif) decreased gambierol sensitivity in Kv3.1 more than 100-fold. Structure determination by homology modeling indicated that the position of the S3b-S4 paddle and its primary structure defines the shape and∖or the accessibility of the binding site for gambierol, explaining the observed differences in gambierol affinity between the channel chimeras. Furthermore, these findings explain the observed difference in gambierol affinity for the closed and open channel configurations of Kv3.1, opening new possibilities for exploring the VSDs as selectivity determinants in drug design.

Modeling conformational transitions in kinases by molecular dynamics simulations: achievements, difficulties, and open challenges.

Protein kinases work because their flexibility allows to continuously switch from inactive to active form. Despite the large number of structures experimentally determined in such states, the mechanism of their conformational transitions as well as the transition pathways are not easily to capture. In this regard, computational methods can help to shed light on such an issue. However, due to the intrinsic sampling limitations, much efforts have been done to model in a realistic way the conformational changes occurring in protein kinases. In this review we will address the principal biological achievements and structural aspects in studying kinases conformational transitions and will focus on the main challenges related to computational approaches such as molecular modeling and MD simulations.