The mechanism of activation of MEK1 by B-Raf and KSR1.

MEK1 interactions with B-Raf and KSR1 are key steps in Ras/Raf/MEK/ERK signaling. Despite this, vital mechanistic details of how these execute signal transduction are still enigmatic. Among these is why, despite B-Raf and KSR1 kinase domains similarity, the B-Raf/MEK1 and KSR1/MEK1 complexes have distinct contributions to MEK1 activation, and broadly, what is KSR1's role. Our molecular dynamics simulations clarify these still unresolved ambiguities. Our results reveal that the proline-rich (P-rich) loop of MEK1 plays a decisive role in MEK1 activation loop (A-loop) phosphorylation. In the inactive B-Raf/MEK1 heterodimer, the collapsed A-loop of B-Raf interacts with the P-rich loop and A-loop of MEK1, minimizing MEK1 A-loop fluctuation and preventing it from phosphorylation. In the active B-Raf/MEK1 heterodimer, the P-rich loop moves in concert with the A-loop of B-Raf as it extends. This reduces the number of residues interacting with MEK1 A-loop, allowing increased A-loop fluctuation, and bringing Ser222 closer to ATP for phosphorylation. B-Raf αG-helix Arg662 promotes MEK1 activation by orienting Ser218 towards ATP. In KSR1/MEK1, the KSR1 αG-helix has Ala826 in place of B-Raf Arg662. This difference results in much fewer interactions between KSR1 αG-helix and MEK1 A-loop, thus a more flexible A-loop. We postulate that if KSR1 were to adopt an active configuration with an extended A-loop as seen in other protein kinases, then the MEK1 P-rich loop would extend in a similar manner, as seen in the active B-Raf/MEK1 heterodimer. This would result in highly flexible MEK1 A-loop, and KSR1 functioning as an active, B-Raf-like, kinase.

The mechanism of activation of monomeric B-Raf V600E.

Oncogenic mutations in the serine/threonine kinase B-Raf, particularly the V600E mutation, are frequent in cancer, making it a major drug target. Although much is known about B-Raf's active and inactive states, questions remain about the mechanism by which the protein changes between these two states. Here, we utilize molecular dynamics to investigate both wild-type and V600E B-Raf to gain mechanistic insights into the impact of the Val to Glu mutation. The results show that the wild-type and mutant follow similar activation pathways involving an extension of the activation loop and an inward motion of the αC-helix. The V600E mutation, however, destabilizes the inactive state by disrupting hydrophobic interactions present in the wild-type structure while the active state is stabilized through the formation of a salt bridge between Glu600 and Lys507. Additionally, when the activation loop is extended, the αC-helix is able to move between an inward and outward orientation as long as the DFG motif adopts a specific orientation. In that orientation Phe595 rotates away from the αC-helix, allowing the formation of a salt bridge between Lys483 and Glu501. These mechanistic insights have implications for the development of new Raf inhibitors.

Clustering and Phase Separation in Mixtures of Dipolar and Active Particles in an External Field.

Directing the assembly of colloidal particles through the use of external electric or magnetic fields shows promise for the creation of reconfigurable materials. Self-propelled particles can also be used to dynamically drive colloidal systems to nonequilibrium steady states. We investigate colloidal systems that combine both of these methods of directed assembly, simulating mixtures of passive dipolar colloids and active soft spheres in an external magnetic field using Brownian dynamics in two dimensions. In these systems, the dipolar particles align in the direction of the external field, but the active particles are unaffected by the field. The phase behaviors exhibited included a percolated dipolar network, dipolar string-fluid, isotropic fluid, and phase-separated state. We find that the external field allows the dipolar particles to form a percolated network more easily compared to when no external field is present. Additionally, the mixture phase separates at lower active particle velocity in an external field than when no field is present. Our results suggest that combining multiple methods of directing colloidal assembly could lead to new pathways to fabricate reconfigurable materials.

Clustering and phase separation in mixtures of dipolar and active particles.

The self-assembly of colloidal particles in dynamic environments has become an important field of study because of potential applications in fabricating out-of-equilibrium materials. We investigate the phase behavior of mixtures of passive dipolar colloids and active soft spheres using Brownian dynamics simulations in two dimensions. The phase behaviors exhibited include dipolar percolated network, dipolar string-fluid, isotropic fluid, and a phase-separated state. We find that the clustering of dipolar colloids is enhanced in the presence of slow-moving active particles compared to the clustering of dipolar particles mixed with passive particles. When the active particle motility is high, the chains of dipolar particles are either broken into short chains or pushed into dense clusters. Motility-induced phase separation into dense and dilute phases is also present. The area fraction of particles in the dilute phase increases as the fraction of active particles in the system decreases, while the area fraction of particles in the dense phase remains constant. Our findings are relevant to the development of reconfigurable self-assembled materials.

Phase diagrams of mixtures of dipolar rods and discs.

Self-assembly of binary mixtures that contain anisotropic, interacting colloidal particles have been proposed as a way to create new, multi-functional materials. We simulate binary mixtures of dipolar rods and dipolar discs in two-dimensions using discontinuous molecular dynamics to determine how the assembled structures of these mixtures differ from those seen in single component systems. Two different binary mixtures are investigated: a mixture of an equal number of dipolar rods and dipolar discs ("equal number"), and a mixture where the area fraction of dipolar rods is equal to the area fraction of dipolar discs ("equal area"). Phase boundaries between fluid, string-fluid, and "gel" phases are calculated and compared to the phase boundaries of the pure components. Looking deeper at the underlying structure of the mixture reveals a complex interplay between the rods and discs and the formation of states where the two components are in different phases. The mixtures exhibit phases where both rods and discs are in the fluid phase, where rods form a string-fluid while discs remain in the fluid phase, a rod string-fluid coexisting with a disc string-fluid, a "gel" that consists primarily of rods while the discs form either a fluid or string-fluid phase, and a "gel" that contains both rods and discs. Our results give insight into the general assembly pathway of binary mixtures, and how complex aggregates can be created by varying the mixture composition, strength of interaction between the two components, and the temperature. By manipulating the properties of one of the components it should be possible to fabricate bifunctional, thermally responsive self-assembled materials.