Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Poster Abstracts

3-POS Board 3 INVESTIGATING THE C-TERMINAL ACTIVATION IN WT AND ONCOGENIC PIK3CA USING ENHANCED SAMPLING SIMULATIONS Zoe Cournia 1 ; Danai Maria Kotzampasi 1 ; 1 Biomedical Research Foundation, Academy of Athens, Athens, Greece PI3K α is the most frequently mutated kinase in human cancers with mutations often occurring at the C-terminus. The C-terminus has a dual function in regulating the kinase, playing an auto inhibitory role for kinase activity and also mediating protein binding to the cell membrane. When PI3K α attaches to the cell membrane, it phosphorylates its substrate PIP2 and converts it to PIP3, initiating a signaling cascade for cell proliferation.[1] Oncogenic mutations in the C-terminus of PI3K α lead to overactivation of the kinase,[2,3] however, the molecular mechanisms by which these C-terminal oncogenic mutations cause PI3K α overactivation remain unclear. H1047R, G1049R, and M1043L mutants increase ATPase activity compared to the WT, and H1047R, G1049R, and a frameshift mutation (N1068KLKR) increase membrane binding compared to the WT.[2,3] Moreover, after comparing available crystal structures of the WT and H1047R mutant, different conformations of the C-terminus are observed with the H1047R mutant structure displaying an open C-terminal conformation associated with PI3K α activation.[4] To understand how C-terminal mutations of PI3K α alter kinase activity, we perform unbiased and biased Molecular Dynamics simulations of the above-mentioned mutants and report the free energy needed for the C-terminal “closed to open” transition and associated conformational changes. H1047R and G1049R mutants have the lowest free energy for the transition compared to the WT, M1043L and N1068KLKR mutants. In the open state of M1043L mutant, a different direction of the C-terminus is observed, which corroborates with experimental work showing that M1043L has a lower membrane-binding ability compared to the other mutants.[3] To validate our findings, we compare the results from Molecular Dynamics simulations with the existing HDX-MS experimental data,[3,5] and find that the results align. The differences in the free energy needed for the closed to open transition and the observed conformational changes across the different mutants provide valuable insights into the molecular mechanisms underlying the C-terminal activation in oncogenic and WT PI3K α . References[1] Kotzampasi DM, Premeti K, Papafotika A, Syropoulou V, Christoforidis S, Cournia Z, et al. The orchestrated signaling by PI3K α and PTEN at the membrane interface. Computational and Structural Biotechnology Journal 2022;20:5607–21. [2] Gkeka P, Evangelidis T, Pavlaki M, Lazani V, Christoforidis S, Agianian B, et al. Investigating the structure and dynamics of the PIK3CA wild-type and H1047R oncogenic mutant. PLoS Comput Biol 2014;10:e1003895. [3] Jenkins ML, Ranga Prasad H, Parson MAH, Harris NJ, Rathinaswamy MK, Burke JE. Oncogenic mutations of PIK3CA lead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus. Nat Commun 2023;14:181. [4] Mandelker D, Gabelli SB, Schmidt-Kittler O, Zhu J, Cheong I, Huang C-H, et al. A frequent kinase domain mutation that changes the interaction between PI3Kalpha and the membrane. Proc Natl Acad Sci U S A 2009;106:16996–7001. [5] Burke JE, Perisic O, Masson GR, Vadas O, Williams RL. Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110 α (PIK3CA). Proc Natl Acad Sci U S A 2012;109:15259–64.

54