Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Poster Abstracts

21-POS Board 21 ELUCIDATING THE INTRICATE REGULATORY MECHANISMS OF RHO GTPASES CANCER ISOFORMS THROUGH COMPUTER SIMULATIONS Angela Parise ; Sofia Cresca 1 ; Alessandra Magistrato 1 ; 1 CNR at SISSA, IOM, Trieste, Italy Rac1 (Ras-related C3 botulinum toxin substrate 1), a member of the Rho-GTPase family, regulates cell adhesion, morphology, and movement, and is over-expressed in various types of cancers. An elegant and intricate system of protein-protein interaction networks regulates the function of Rho-GTPases. GTPase-activating proteins (GAPs) and guanine exchange factors (GEFs) repeatedly promote the exchange between GTP and GDP, thereby allowing the switch between the active and inactive forms of the Rho GTPase.The cryo-EM structures of DOCK proteins and their small-GTPase complexes have provided insights into the GEF activity mechanism. However, the regulatory mechanism of DOCKs by partner proteins and the signal transduction events responsible for Rho-GTPase regulation in larger macromolecular assemblies remain elusive. Due to their key regulatory role, Rho GTPases are the object of cancer-associated mutations, and following the recent approval of Sotorasib, they are emerging as amenable targets for the discovery of small molecule covalent inhibitors.In this contribution, the regulatory mechanisms of Rac1 are revealed by all-atom simulations. Several model systems are compared, starting with the homodimeric cryo-EM structure of ELMO1-DOCK5-Rac1 (1). The monomer and the DOCK5-Rac1 complex bound to GDP and in the apo form are also investigated. Additionally, a comparison between the different wild-type systems, the most frequent Rac1P29 mutations, and the Rac1b splice variant is done. Different types of simulations reveal: (i) the molecular details of the main functional motifs of Rac1 and Rac1 pathological isoforms; (ii) the structural and dynamical features of ELMO1-DOCK5-Rac1 complexes; (iii) the GTP binding affinities and the residence time of the GDP product within the catalytic pocket of wild-type and Rac1 cancer isoforms [2]; and (iv) ELMO1-DOCK5-Rac1 membrane interactions.(1)Kukimoto Niino, Mutsuko, et al. "Cryo-EM structure of the human ELMO1-DOCK5-Rac1 complex." Science advances 7.30 (2021): eabg3147.(2)Parise, Angela, and Alessandra Magistrato. "Assessing the mechanism of fast‐cycling cancer‐associated mutations of Rac1 small Rho GTPase." Protein Science 33.4 (2024): e4939.

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