The Effect of a Widespread Cancer-Causing Mutation on the Inactive to Active Dynamics of the B‑Raf Kinase

Protein kinases play a key role in regulating cellular processes. Kinase dysfunction can lead to disease, making them an attractive target for drug design. The B-Raf kinase is a key target for the treatment of melanoma since a single mutation (V600E) is found in more than 50% of all malignant melanomas. Despite the importance of B-Raf in melanoma treatment, the molecular mechanism by which the mutation increases kinase activity remains elusive. Since kinases are tightly regulated by a conformational transition between an active and inactive state, which is difficult to capture experimentally, large-scale enhanced-sampling simulations are performed to examine the mechanism by which the V600E mutation enhances the activity of the B-Raf monomer. The results reveal that the mutation has a twofold effect. First, the mutation increases the barrier of the active to inactive transition trapping B-Raf in the active state. The mutation also increases the flexibility of the activation loop which might speed-up the rate-limiting step of phosphorylation. Both effects can be explained by the formation of salt-bridges with the Glu600 residue

Conformational Changes and Free Energies in a Proline Isomerase

Proteins are dynamic molecules and their ability to adopt alternative conformations is central to their biological function. The structural and biophysical properties of transiently and sparsely populated states are, however, difficult to study and an atomic-level description of those states is challenging. We have used enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from X-ray crystallography and NMR, to describe quantitatively the transition between the major and a minor state of Cyclophilin A, thus providing new insight into how dynamics can affect enzyme function. We calculate the conformational free energy between the two states, and comparison with experiments demonstrates a surprisingly high accuracy for both the wild type protein and a mutant that traps the protein in its alternative conformation. Our results demonstrate how the combination of state-of-the-art force fields and enhanced sampling methods can provide a detailed and quantitative description of the conformational changes in proteins such as those observed in Cyclophilin A

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

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

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

List of c-Abl point mutants investigated in this study with summary of the effect of mutations.

<p>Mutants were tested for c-Abl activity via immunoblotting of HEK293 lysates or immunoprecipitates (IP) and via kinase activity assay. The numbering of residues is in agreement with the sequence of the isoform Ib. Activity scoring (effect of the mutation):, nt, not tested, − inactive (mutation disruptive), + weakly active (mutation mildly disruptive), ++ activity similar to wild-type (mutation neutral), +++ hyperactive (mutation activating) (See also Supplemental Table S1).</p><p>List of c-Abl point mutants investigated in this study with summary of the effect of mutations.</p

Domain organization and crystal structures of Abl kinase.

<p><b>A</b> The c-Abl isoform Ib is characterized by myristoylation (Myr) on Gly-2 of the N-terminal capping region (cap). The tyrosine kinase domain is preceded by the SH3 and SH2 domains and a connecting linker. The last exon region contains nuclear localization signals and a C-terminal actin binding domain (ABD). <b>B</b> In the down-regulated state (PDB entry 2FO0), the SH2 domain binds the C-lobe of the kinase domain, the myristate is bound in its cognate pocket and the SH3 domain binds the SH2-CD linker. <b>C</b> In the active “top-hat” conformation (PDB entry 1OPL), the SH2 domain moves to interact with the N-lobe of the kinase domain. The αC helix and the activation loop are highlighted in red and pink, respectively. <b>D</b> Positions of the most important point mutations at the SH2-CD interface and in the β3-αC loop.</p

Effect of point mutations on the kinase activity of c-Abl.

<p>Abl proteins were immunoprecipitated and assayed for phosphorylation of an optimal Abl substrate peptide. The kinase activity was normalized to the amount of protein and to the activity of the wild-type CD or SH2-CD construct. <b>A</b> M297L was found to decrease kinase activity, whereas M297G had an activating effect in the absence of the SH2 domain. The kinase-dead mutant D382N presented no activity, and the known inactivating (I164E) and activating (T231R) mutations also show, accordingly, a decreased and increased activity. <b>B</b> The M297G mutant is significantly more active in the context of the isolated kinase domain, but not in the presence of the SH2 domain. The error bars are standard deviations from biological quadruplicates (n = 4, **P<0.01, Student <i>t</i> test). <b>C</b> Mutations E294P and E294P V299P have an activating effect. The reactions were performed at 37°C. <b>D</b> The activity of wild-type and E294P V299P Abl proteins was measured at increasing substrate concentrations and 25 µM ATP. <b>E</b> Y339G substitution is neutral, whereas Y339P diminishes Abl activity. The effect is seen both at 24°C and at an elevated temperature. <b>F, G</b> The ratio of the kinase activity at the elevated and room temperatures. Except for Y339P, the SH2-CD proteins retain their activity at the elevated temperature, whereas the activity of the CD constructs is reduced at least 2-fold. The error bars are standard deviations from technical triplicates except for (B). (See also Figures S4 and S5).</p

The free energy of conformational changes of the A-loop.

<p>The free energy surfaces of the A-loop transition from open (active-like) to closed (inactive-like) conformation in case of the ABL catalytic domain alone (left) and in presence of the SH2 regulatory domain in the “top-hat” conformation as a function of the contact map distances to the respective reference structures. For the deepest minima a representative structure is also shown below with the CD colored in blue, the SH2 in green, the A-loop in yellow and the aC-helix in red.</p

Allosteric coupling and flexibility of c-Abl.

<p><b>A</b> Allosteric couplings of residues in the CD to the SH2 domain. High values (yellow and red) indicate strong allosteric interactions (See also Supplemental <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003863#pcbi.1003863.s001" target="_blank">Figure S1</a> A–C). <b>B</b> Free CD colored by RMSF from MD simulations. <b>C</b> SH2-CD construct colored by RMSF. (See also <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003863#pcbi.1003863.s002" target="_blank">Figure S2</a>).</p