Pattern and Dynamics of FLT3 Duplications.

FMS-like tyrosine kinase 3 (FLT3) is mutated in ∼30% of patients that suffer from acute myeloid leukemia (AML). In about 25% of all AML patients, in-frame insertions are observed in the sequence. Most of those insertions are internal tandem duplications (ITDs) of a sequence from the protein. The characteristics of such mutations in terms of length, sequence, and location were hitherto studied in different populations, but not in a comprehensive mutation database. Here, in-frame insertions into the FLT3 gene were extracted from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. These were analyzed with respect to the length, location, and sequence of the mutations. Furthermore, characteristic strings (sequences) of different lengths were identified. Mutations were shown to occur most often in the juxtamembrane zipper (JM-Z) domain of FLT3, followed by the hinge domain and first tyrosine kinase domain (TKD1), upstream of the phosphate-binding loop (P-loop). Interestingly, there are specific hot spot residues where insertions are more likely to occur. The insertions vary in length between one and 67 amino acids, with the largest insertions spanning the phosphate binding loop. Insertions that occur downstream of the P-loop are shorter. Our analysis further shows that acidic and aromatic residues are enriched in the insertions. Finally, molecular dynamics simulations were run for FLT3 with ITD insertions in the hinge and tyrosine kinase domains. On the basis of the findings, a mechanism is proposed for activation by ITDs, according to which there is no direct coupling between the length of the insertion and the activity of the mutated protein. The effect of insertions on the sensitivity of FLT3 to kinase inhibitors is discussed based on our findings.

Conformational modifications induced by internal tandem duplications on the FLT3 kinase and juxtamembrane domains.

The aberrant expression of FLT3 tyrosine kinase is associated primarily with acute myeloid leukaemia. This blood malignancy is often related to the onset of internal tandem duplications (ITDs) in the native sequence of the protein. The ITDs occur mainly in the juxtamembrane domain of the protein and alter the normal activity of the enzyme. In this work, we have studied the native form of FLT3 and six mutants by molecular dynamics simulations. The catalytic activity of FLT3 is exerted by the tyrosine kinase domain (KD) and regulated by the juxtamembrane (JM) domain. Analysis of the dynamics of these two domains have shown that the introduction of ITDs in the JM domain alters both structural and dynamic parameters. The presence of ITDs allowed the protein to span a larger portion of the conformational space, particularly in the JM domain and the activation loop. The FLT3 mutants were found to adopt more stable configurations than the native enzyme. This was due to the different arrangements assumed by the JM domain. Larger fluctuations of the activation loop were found in four of the six mutants. In the native FLT3, the key residue Tyr572 is involved in a strong and stable interaction with an ion pair. This interaction, which is thought to keep the JM in place hence regulating the activity of the enzyme, was found to break in all FLT3 mutants.

Activation and Inactivation of the FLT3 Kinase: Pathway Intermediates and the Free Energy of Transition.

The aberrant expression of kinases is often associated with pathologies such as cancer and autoimmune diseases. Like other types of enzymes, kinases can adopt active and inactive states, where a shift toward more stable active state often leads to disease. Dozens of kinase inhibitors are, therefore, used as drugs. Most of these bind to either the inactive or active state. In this work, we study the transitions between these two states in FLT3, an important drug target in leukemias. Kinases are composed of two lobes (N- and C-terminal lobes) with the catalytic site in-between. Through projection of the largest motions obtained through molecular dynamics (MD) simulations, we show that each of the end-states (active or inactive) already possess the ability for transition as the two lobes rotate which initiates the transition. A targeted simulation approach known as essential dynamics sampling (EDS) was used to speed up the transition between the two protein states. Coupling the EDS to implicit-solvent MD was performed to estimate the free energy barriers of the transitions. The activation energies were found in good agreement with previous estimates obtained for other kinases. Finally, we identified FLT3 intermediates that assumed configurations that resemble that of the c-Src nonreceptor tyrosine kinase. The intermediates show better binding to the drug ponatinib than c-Src and the inactive state of FLT3. This suggests that targeting intermediate states can be used to explain the drug-binding patterns of kinases and for rational drug design.

The catalytic activity of Abl1 single and compound mutations: Implications for the mechanism of drug resistance mutations in chronic myeloid leukaemia.

BACKGROUND: Abl1 is a protein tyrosine kinase whose aberrant activation due to mutations is the culprit of several cancers, most notably chronic myeloid leukaemia. Several Abl1 inhibitors are used as anti-cancer drugs. Unfortunately, drug resistance limits their effectiveness. The main cause for drug resistance is mutations in the kinase domain (KD) of Abl1 that evolve in patients. The T315I mutation confers resistance against all clinically-available inhibitors except ponatinib. Resistance to ponatinib can develop by compound (double) mutations. METHODS: Kinetic measurements of the KD of Abl1 and its mutants were carried out to examine their catalytic activity. Specifically, mutants that lead to drug resistance against ponatinib were considered. Molecular dynamics simulations and multiple sequence analysis were used for explanation of the experimental findings. RESULTS: The catalytic efficiency of the T315I pan-resistance mutant is more than two times lower than that of the native KD. All ponatinib resistant mutations restore the catalytic efficiency of the enzyme. Two of them (G250E/T315I and Y253H/E255V) have a catalytic efficiency that is more than five times that of the native KD. CONCLUSIONS: The measurements and analysis suggest that resistance is at least partially due to the development of a highly efficient kinase through subsequent mutations. The simulations highlight modifications in two structurally important regions of Abl1, the activation and phosphate binding loops, upon mutations. GENERAL SIGNIFICANCE: Experimental and computational methods were used together to explain how mutations in the kinase domain of Abl1 lead to resistance against the most advanced drug currently in use to treat chronic myeloid leukaemia.

Role of Stone-Wales defects on the interfacial interactions among graphene, carbon nanotubes, and Nylon 6: A first-principles study.

We investigate computationally the role of Stone-Wales (SW) defects on the interfacial interactions among graphene, carbon nanotubes (CNTs), and Nylon 6 using density functional theory (DFT) and the empirical force-field. Our first-principles DFT calculations were performed using the Quantum ESPRESSO electronic structure code with the highly accurate van der Waals functional (vdW-DF2). Both pristine and SW-defected carbon nanomaterials were investigated. The computed results show that the presence of SW defects on CNTs weakens the CNT-graphene interactions. Our result that CNT-graphene interaction is much stronger than CNT-CNT interaction indicates that graphene would be able to promote the dispersion of CNTs in the polymer matrix. Our results demonstrate that carbon nanomaterials form stable complexes with Nylon 6 and that the van der Waals interactions, as revealed by the electronic charge density difference maps, play a key stabilizing role on the interfacial interactions among graphene, CNTs, and Nylon 6. Using the density of states calculations, we observed that the bandgaps of graphene and CNTs were not significantly modified due to their interactions with Nylon 6. The Young's moduli of complexes were found to be the averages of the moduli of their individual constituents.

A mechanochemical study of the effects of compression on a Diels-Alder reaction.

We examine the effects of compressive external forces on the mechanisms of the parent Diels-Alder (DA) reaction between butadiene and ethylene. Reaction pathways and transition states were calculated using the nudged elastic band method within a mechanochemical framework at the CASSCF(6,6)/6-31G**, as well as the B3LYP/6-311++G** levels of theory. Our results suggest that compressive hydrostatic pressure lowers the energy barrier for the parent DA reaction while suppressing the undesirable side reaction, thereby leading to a direct increase in the yield of cyclohexene. Compressive pressure also increases the exothermicity of the parent DA reaction, which would lead to increased temperatures in a reaction vessel and thereby indirectly increase the yield of cyclohexene. Our estimates indicate that the compression used in our study corresponds to a range of 68 MPa-1410 MPa.

Influence of mutations at the proximal histidine position on the Fe-O2 bond in hemoglobin from density functional theory.

Four mutated hemoglobin (Hb) variants and wild type hemoglobin as a reference have been investigated using density functional theory methods focusing on oxygen binding. Dispersion-corrected B3LYP functional is used and found to provide reliable oxygen binding energies. It also correctly reproduces the spin distribution of both bound and free heme groups as well as provides correct geometries at their close vicinity. Mutations in hemoglobin are not only an intrigued biological problem and it is also highly important to understand their effects from a clinical point of view. This study clearly shows how even small structural differences close to the heme group can have a significant effect in reducing the oxygen binding of mutated hemoglobins and consequently affecting the health condition of the patient suffering from the mutations. All of the studied mutated Hb variants did exhibit much weaker binding of molecular oxygen compared to the wild type of hemoglobin.

Influence of antifreeze proteins on the ice/water interface.

Antifreeze proteins (AFP) are responsible for the survival of several species, ranging from bacteria to fish, that encounter subzero temperatures in their living environment. AFPs have been divided into two main families, moderately and hyperactive, depending on their thermal hysteresis activity. We have studied one protein from both families, the AFP from the snow flea (sfAFP) and from the winter flounder (wfAFP), which belong to the hyperactive and moderately active family, respectively. On the basis of molecular dynamics simulations, we have estimated the thickness of the water/ice interface for systems both with and without the AFPs attached onto the ice surface. The calculation of the diffusion profiles along the simulation box allowed us to measure the interface width for different ice planes. The obtained widths clearly show a different influence of the two AFPs on the ice/water interface. The different impact of the AFPs here studied on the interface thickness can be related to two AFPs properties: the protein hydrophobic surface and the number of hydrogen bonds that the two AFPs faces form with water molecules.

Induced ice melting by the snow flea antifreeze protein from molecular dynamics simulations.

Antifreeze proteins (AFP) allow different life forms, insects as well as fish and plants, to survive in subzero environments. AFPs prevent freezing of the physiological fluids. We have studied, through molecular dynamics simulations, the behavior of the small isoform of the AFP found in the snow flea (sfAFP), both in water and at the ice/water interface, of four different ice planes. In water at room temperature, the structure of the sfAFP is found to be slightly unstable. The loop between two polyproline II helices has large fluctuations as well as the C-terminus. Torsional angle analyses show a decrease of the polyproline II helix area in the Ramachandran plots. The protein structure instability, in any case, should not affect its antifreeze activity. At the ice/water interface the sfAFP triggers local melting of the ice surface. Bipyramidal, secondary prism, and prism ice planes melt in the presence of AFP at temperatures below the melting point of ice. Only the basal plane is found to be stable at the same temperatures, indicating an adsorption of the sfAFP on this ice plane as confirmed by experimental evidence.

Glucose oxidase from Penicillium amagasakiense: characterization of the transition state of its denaturation from molecular dynamics simulations.

Glucose oxidase (GOx) is a flavoenzyme having applications in food and medical industries. However, GOx, as many other enzymes when extracted from the cells, has relatively short operational lifetimes. Several recent studies (both experimental and theoretical), carried out on small proteins (or small fractions of large proteins), show that a detailed knowledge of how the breakdown process starts and proceeds on molecular level could be of significant help to artificially improve the stability of fragile proteins. We have performed extended molecular dynamics (MD) simulations to study the denaturation of GOx (a protein dimer containing nearly 1200 amino acids) to identify weak points in its structure and in this way gather information to later make it more stable, for example, by mutations. A denaturation of a protein can be simulated by increasing the temperature far above physiological temperature. We have performed a series of MD simulations at different temperatures (300, 400, 500, and 600 K). The exit from the protein's native state has been successfully identified with the clustering method and supported by other methods used to analyze the simulation data. A common set of amino acids is regularly found to initiate the denaturation, suggesting a moiety where the enzyme could be strengthened by a suitable amino acid based modification.

Inclusion of chloromethane guests affects conformation and internal dynamics of cryptophane-D host.

Cryptophane-D is composed of two nonequivalent cyclotribenzylene caps bound together by three OCH2CH2O bridges in a syn arrangement. Host-guest complexes with chloroform and dichloromethane were investigated in solution by NMR spectroscopy. Variable temperature NMR (1)H and (13)C spectra showed effects of chemical exchange between the free and bound guest and of conformational exchange for the host, strongly and specifically affected by guest binding. We found in particular that the carbon-13 chemical shifts for the linkers connecting the two cyclotribenzylene units are very informative. The NMR results were supported by DFT calculations. The guest exchange was also studied quantitatively, either by EXSY measurements (for chloroform as guest) or by line-shape analysis (for dichloromethane as guest). In the case of chloroform guest, we also investigated cross-relaxation between the guest and host protons, as well as carbon-13 longitudinal relaxation and heteronuclear NOE at three different fields. The results were interpreted in terms of orientation and dynamics of the guest inside the host cavity. Putting together various types of evidence resulted in remarkably detailed insight into the process of molecular recognition of the two guests by cryptophane-D host.