Ozguney, B. | Search bioRxiv

bioRxiv2025

Mutation-Induced Effects on Rac1 Conformational Dynamics: Implications for Therapeutic Targeting

Understanding the conformational dynamics of proteins, particularly small GTPases like Rac1, is vital for elucidating their functional mechanisms and developing targeted therapies. Rac1, pivotal in cellular processes, toggles between inactive GDP-bound and active GTP-bound states, regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Mutations, such as Rac1s spontaneously activating oncogenic gain-of-function mutation P29S, associated with cancer, disrupt this equilibrium, leading to aberrant signaling. Traditional drug targeting of Rac1 is challenging due to its biological complexity and the lack of accessible active sites on its surface, necessitating alternative strategies. We propose a computational framework integrating Molecular Dynamics (MD) simulations and Elastic Network Models (ENM) to explore conformational dynamics. Our findings highlight the interplay between Mg2+ binding and conformational ensembles, revealing enhanced conformational heterogeneity in both inactive and active states upon P29S mutation. The critical location of P29S, Mg2+ coordination site, and GDP/GTP binding pocket with respect to global hinges provides mechanistic insight into how this mutation disrupts normal protein function through altered metal coordination dynamics. Furthermore, we identified strategic positions as potential "rescue mutation" sites, with T75A showing particular promise in mitigating the destabilizing conformational effects of P29S. Overall, this work provides insights into Rac1s dynamic behavior and offers a foundation for targeted drug design strategies.

biophysics↗

bioRxiv2024

RNA-binding tunes the conformational plasticity and intradomain stability of TDP-43 tandem RNA recognition motifs

TAR DNA binding protein 43 (TDP-43) is a nuclear RNA/DNA-binding protein with pivotal roles in RNA-related processes such as splicing, transcription, transport, and stability. The high binding affinity and specificity of TDP-43 towards its cognate RNA sequences (GU-rich) is mediated by highly conserved residues in its tandem RNA recognition motif (RRM) domains (aa:104-263). Importantly, the loss of RNA-binding to the tandem RRMs caused by physiological stressors and chemical modifications promotes cytoplasmic mislocalization and pathological aggregation of TDP-43. Despite the substantial implications of RNA in TDP-43 function and pathology, a comprehensive characterization of the effect of RNA-binding on conformational dynamics, interdomain interactions and intradomain stability of the tandem RRMs has not yet been conducted. Here, we employed all-atom molecular dynamics (MD) simulations to assess the effect of RNA-binding on the conformational landscape and intradomain stability of TDP-43 tandem RRMs. Our simulations reveal a high intrinsic conformational plasticity of the tandem RRMs in the absence of RNA which surprisingly, is accompanied by a tendency of RRM1 to adopt partially-unfolded conformations. While binding to RNA limits the overall conformational space of the tandem RRMs and promotes intradomain stability, several RRM-RNA contacts mediated by highly conserved residues are observed to be far more dynamic than previously inferred from NMR structural ensemble. Overall, our simulations reveal how RNA dynamically tunes the structural and conformational landscape of TDP-43 tandem RRMs, contributing to physiological function and mitigating pathological aggregation. SIGNIFICANCEThe cytoplasmic mislocalization and aggregation of TDP-43 due to loss of its RNA-binding capability is associated with the onset and progression of neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Due to the flexible nature of RNA and the presence of a disordered linker between RRM domains, characterizing the dynamic interactions between RRMs-RNA and/or RRM1-RRM2 by experiments alone has remained challenging. In this study, we performed all-atom simulations initiated from the NMR conformers of RNA-bound tandem RRMs of TDP-43 to investigate their underlying structural and conformational dynamics. Our findings indicate that RNA binding effectively reduces conformational heterogeneity in the tandem RRMs and acts as a protective factor for the unfolding and aggregation of RRM1. These effects are achieved through a combination of stable and dynamic protein-RNA interactions which involve highly conserved amino acids.

biophysics↗