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.
Highlights
Rap belongs to the family of small Ras-like GTPases, which have many roles in cellular activities like cell proliferation, apoptosis and differentiation etc.[1,2,3,4,5]
We explore the effects of such phosphorylation on the dynamics of functional loops such as Switch I and Switch II loops to characterize the allosteric pathways within Rap-Raf complex and subsequently gain some insight into possible mechanisms through which Rap may affect the downstream MAPK signalling pathway
The Solvent accessible surface area (SASA) of SER11 residue calculated for the molecular dynamics trajectory of Raf-Rap complex with GTP indicates that the dynamical nature of the P-loop allows the buried phosphosite SER11 to be exposed to water for a significant time on the present simulation timescale, suggesting a strong possibility of a kinase phosphorylating the SER11 residue and lends validity to our simulation studies involving phosphorylated Rap protein (Fig. S3)
Summary
Rap belongs to the family of small Ras-like GTPases, which have many roles in cellular activities like cell proliferation, apoptosis and differentiation etc.[1,2,3,4,5]. The Ras GTPases participate in many signalling pathways, including the MAPK/ERK, PI3K7–9 Many factors including their cellular location, bound ligand molecule and phosphorylation can affect how these molecules interact with downstream signalling proteins[10,11], which is crucial in transmitting signal from Ras to the mitogen-activated protein kinase. Not as well studied as the role of GEF proteins and GAP catalyzed changes in the nucleotide bound conformations, phosphorylation is known to regulate the functions of Ras and Rap proteins[28,29,30,31,32]. We study the effects of phosphorylation of a single residue, SER11, identified as a possible phosphosite in Rap1A (Supplemental Table 1 of 37 and Table S1 of 49) (see Fig. 1), on the conformational dynamics of the Rap1A and its interactions with the effector protein kinase c-Raf[1]. We explore the effects of such phosphorylation on the dynamics of functional loops such as Switch I and Switch II loops to characterize the allosteric pathways within Rap-Raf complex and subsequently gain some insight into possible mechanisms through which Rap may affect the downstream MAPK signalling pathway
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