Phosphorylation promotes binding affinity of Rap-Raf complex by allosteric modulation of switch loop dynamics

Abstract

The effects of phosphorylation of a serine residue on the structural and dynamic properties of Ras-like protein, Rap, and its interactions with effector protein Ras binding domain (RBD) of Raf kinase, in the presence of GTP, are investigated via molecular dynamics simulations. The simulations show that phosphorylation significantly effects the dynamics of functional loops of Rap which participate in the stability of the complex with effector proteins. The effects of phosphorylation on Rap are significant and detailed conformational analysis suggest that the Rap protein, when phosphorylated and with GTP ligand, samples different conformational space as compared to non-phosphorylated protein. In addition, phosphorylation of SER11 opens up a new cavity in the Rap protein which can be further explored for possible drug interactions. Residue network analysis shows that the phosphorylation of Rap results in a community spanning both Rap and RBD and strongly suggests transmission of allosteric effects of local alterations in Rap to distal regions of RBD, potentially affecting the downstream signalling. Binding free energy calculations suggest that phosphorylation of SER11 residue increases the binding between Rap and Raf corroborating the network analysis results. The increased binding of the Rap-Raf complex can have cascading effects along the signalling pathways where availability of Raf can influence the oncogenic effects of Ras proteins. These simulations underscore the importance of post translational modifications like phosphorylation on the functional dynamics in proteins and can be an alternative to drug-targeting, especially in notoriously undruggable oncoproteins belonging to Ras-like GTPase family.

Figure 1

Rap-Raf protein complex (crystal structure, PDB ID 1C1Y) showing the location of important functional loops like P-loop, Switch I, Switch II and RBD loop regions. The phosphosite SER11 is shown in green.

Figure 2

The RMSF of residues of Rap C_α atoms averaged over 350 to 400 ns of simulation for both unphosphorylated (green) and phosphorylated (blue). The relevant Switch I and Switch II loops are indicated in the figure.

Figure 3

Overlapped snapshots of GTP bound Rap protein showing the conformation of functional Switch I and Switch II loops with (red) and without (yellow) phosphorylation towards the end of the simulation. The positions of residues GLY 12 and GLY60 with (red) and without(yellow) phosphorylation is also marked.

Figure 4

The time evolution of (a) distance between GLY60(O) and GLY12(N) atoms, (b) distance between GLY60(center of mass) and GTP(O3G) atoms for both unphosphorylated (green) and phosphorylated (blue) cases.

Figure 5

Snapshots of nucleotide pocket of Rap-GTP with and without phosphorylation of SER11. The functional loops Switch I and Switch II loops are shown in red and yellow colour respectively. The relevant residues in the two loops are also shown and the hydrogen bonds between GTP and residues in Switch I loop, when relevant, are shown in white dashed lines.

Figure 6

The time evolution of distance between the catalytic residue THR61 and GTP ligand without (green) and with (blue) phosphorylation.

Figure 7

Cross-correlation plots of the Rap complex for 300 to 400 ns trajectory data of GTP- and GTP-PSER11 cases.

Figure 8

Conformer plots of Rap and Raf domains respectively(375 to 400 ns simulation data). The plot shows conformational space sampled by Rap protein in terms of PC1(80.13%) and PC2(2.36%).

Figure 9

Largest cavity location of Rap domain in original crystal structure and last 5 ns averaged structures of GTP (red; volume: 605.96 ų), Crystal structure(blue; volume: 521.01 ų) and GTP-PSER11(black; 278.01 ų) cases.

Figure 10

The time evolution of (a) distance between Rap:TYR32(CA) and Raf:LYS84(CA) atoms, (b) distance between Rap:ASP38(OD1) and Raf:THR68(OG1) atoms.

Figure 11

The communities detected in (a) GTP- and (b) GTP-PSER11 cases. The yellow community represents the L4 loop.