Summary of the fixed-protonation-state MD simulations (aggregate time of 135 μs)

The X-ray structure of the BRAFV600E dimer in complex with PHI1.

Left. Cartoon representation of the BRAFV600E dimer in complex with PHI1 (PDB: 6P7G Cotto-Rios et al. (2020), two protomers are colored tan and grey). The αC-helix, a-loop, and c-loop are colored orange, yellow, and pink, respectively. Right. A zoomed-in view of a PHI1-bound protomer. PHI1 and the sidechains of DFG-Asp594, αC-Glu501, catalytic Lys483, and HRD-His574 are shown as sticks.

Protein-ligand interaction fingerprints for PHI1, LY3009120, Vermurafenib, and the inhibition of ERK1/2 phosphorylation in melanoma cells.

a) Left. Visualization of the back pockets (BPs) in BRAFV600E in complex with PHI1. BP-I, BP-II, and BP-III are colored blue, orange, and green, respectively. BP definitions of Liao (Liao, 2007) are followed. a) Right. Chemical structures of the example dimer selective (PHI1), equipotent (LY3009120 or LY), and monomer selective (Vermurafenib or VEM) inhibitors of BRAFV600E. Portions of structures are highlighted according to the BPs they occupy in the co-crystal structure (PDB IDs: 6P7G, 5C9C, and 4RZV). b) Protein-ligand interaction fingerprints for PHI1, LY, and VEM in BRAFV600E according to the co-crystal structures (PDB IDs: 6P7G, 5C9C, and 4RZV). White indicates no interaction, while grey, blue, and red indicate hydrophobic, h-bond donor (H-donor) and acceptor (H-acceptor) interactions, respectively. These interactions were calculated by KLIFS (Kooistra et al., 2016) and manually verified and corrected. A h-bond was defined using the donor-accept distance cutoff of 3.5 Å, and a hydrophobic contact cutoff of 4 Å was used for aromatic interactions and 4.5 Å for non-aromatic interactions. For simplicity, aromatic face-to-face interactions are indicated as hydrophobic. An extensive list of monomer-selective and dimer-compatible inhibitors with co-crystal structures is given in Supplemental Table 1. c,d) Inhibition of ERK1/2 T202/Y204 phosphorylation in SKMEL239 (c) and SKMEL239-C4 (d) melanoma cells (50,000 cells/well) following one hour treatment at 37°C by PHI1, LY3009120, and Vemurafenib in different concentrations. Normalized values and non-linear regression fits of ERK phosphorylation % are shown for different compounds. Error bars represent mean±SEM with n=3.

Dimerization and inhibitor binding modulate the conformation and dynamics of the αC-helix and DFG motif of BRAFV600E.

a-f) Probability distribution of the αC position, probability of the Lys483–Glu501 salt bridge, and probability distribution of the DFG pseudo dihedral angle in the apo monomer (blue), apo dimer (orange), PHI1-bound dimer (green), and LY-bound dimer BRAFV600E. The αC position is defined by the distance between the Cα of Ile582 on β7 and the Cα center of mass of Asn500, Glu501, and Val502 (Kanev et al., 2020). A salt bridge between Lys483 and Glu501 is defined by a cutoff distance of 4 Å between the nitrogen of Lys483 and the nearest carboxylate oxygen of Glu501; the standard deviation of the probability across replicas are shown as error bars. The DFG pseudo dihedral is defined by the Cα atoms of Ile592, Gly593, Asp594, and Phe595 (Möbitz, 2015). g-j) Density plots of the αC position vs. the minimum distance between Glu501 and the amide group of PHI1 (g,i) or LY (h,j) in the holo dimer (g,h) or holo monomer (i,j) BRAFV600E.

Both PHI1 and LY stabilize the interprotomer contacts of BRAFV600E.

Left. The N-lobe (blue for A; grey for B) and C-lobe (red for A; orange for B) of each protomer in the BRAFV600E dimer are separated into different communities according to the difference contact network analysis (Yao et al., 2018). Right. The average number of interprotomer contacts was calculated for the apo and holo BRAFV600E dimer. (PHI1 top or LY(bottom)). The difference between the holo and apo contacts is shown in the graph form for PHI1 (top) and LY (bottom), and the sum (0.3) is given. Interprotomer contacts are shown as blue (more contacts in holo simulations) or red (more contacts in apo simulations) edges. The difference contact network analysis was performed using the dCNA program (Yao et al., 2018). The cutoff distance defining a contact was 4.5 Å; the threshold for determining a stable contact was set to 0.7, and the number of communities was set to 4.

Conformation of theαC helix and DFG motif is dependent on the presence or absence of PHI1 in the second protomer.

a) The he Glu501–Lys483 salt bridge, and DFG pseudo dihedral of the apo protomer in the one PHI1- (blue) or one simulations. As a reference, the apo dimer data is shown in grey. b) The same quantities as in a) but for the holo or LY-bound (orange) mixed dimer simulations. As a reference, the two PHI1- and LY-bound holo dimer data pectively. The standard deviation of the probability across replicas is shown in error bars. c) Snapshot from both PHI1- (cyan) and LY-bound (orange) holo protomers (gray). The αC-helix of the apo protomer is highlighted in for LY-bound mixed dimer. For simplicity, only the apo protomer from the PHI1-bound mixed dimer is shown.

A working model that explains dimer selectivity and binding cooperativity of BRAFV600E inhibitors.

Top left. In the monomeric BRAFV600E, the αC-helix (orange) is very flexible and exclusively samples the out states. Top right. Upon dimerization, the αC-helix is restrained and shifts inward, while the DFG-motif maintains its conformation but gains significant flexibility. Bottom right. When the first PHI1 molecule binds, its amide linker donates a h-bond to the carboxylate of Glu501 (orange stick) in the first protomer, which locks the αC helix to the αC-in state; it also shifts and restricts the DFG-motif into the DFG-out state through the interaction with the DFG-Asp backbone. The αC-helix and DFG-motif in the second unbound protomer are also affected, with the αC-helix shifting towards αC-in while DFG-motif (slightly) moving towards DFG-out; these conformational changes are in the direction of the inhibitor-bound state. Thus, the allosteric pre-organization primes the second protomer for accepting a second PHI1 molecule. Bottom left. When the second PHI1 molecule binds, the αC helix and DFG-motif in both protomers are shifted and fully locked into the αC-in and DFG-out states.