Multiscale simulations reveal the driving forces of p53C phase separation accelerated by oncogenic mutations.

Liquid-Liquid phase separation (LLPS) of p53 to form liquid condensates has been implicated in cellular functions and dysfunctions. The p53 condensates may serve as amyloid fibril precursors to initiate p53 aggregation, which is associated with oncogenic gain-of-function and various human cancers. M237I and R249S mutations located in p53 core domain (p53C) have been detected respectively in glioblastomas and hepatocellular carcinoma. Interestingly, these p53C mutants can also undergo LLPS and liquid-to-solid phase transition, which are faster than wild type p53C. However, the underlying molecular basis governing the accelerated LLPS and liquid-to-solid transition of p53C remain poorly understood. Herein, we explore the M237I/R249S mutation-induced structural alterations and phase separation behavior of p53C by employing multiscale molecular dynamics simulations. All-atom simulations revealed conformational disruptions in the zinc-binding domain of the M237I mutant and in both loop3 and zinc-binding domain of the R249S mutant. The two mutations enhance hydrophobic exposure of those regions and attenuate intramolecular interactions, which may hasten the LLPS and aggregation of p53C. Martini 3 coarse-grained simulations demonstrated spontaneous phase separation of p53C and accelerated effects of M237I/R249S mutations on the phase separation of p53C. Importantly, we find that the regions with enhanced intermolecular interactions observed in coarse-grained simulations coincide with the disrupted regions with weakened intramolecular interactions observed in all-atom simulations, indicating that M237I/R249S mutation-induced local structural disruptions expedite the LLPS of p53C. This study unveils the molecular mechanisms underlying the two cancer-associated mutation-accelerated LLPS and aggregation of p53C, providing avenues for anticancer therapy by targeting the phase separation process.

Oncogenic R248W mutation induced conformational perturbation of the p53 core domain and the structural protection by proteomimetic amyloid inhibitor ADH-6.

The involvement of p53 aggregation in cancer pathogenesis emphasizes the importance of unraveling the mechanisms underlying mutation-induced p53 destabilization. And understanding how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations is pivotal for the development of therapeutics targeting p53-aggregation-associated cancers. A recent experimental study highlights the efficacy of the proteomimetic amyloid inhibitor ADH-6 in stabilizing R248W p53 and inhibiting its aggregation in cancer cells by interacting with the p53 core domain (p53C). However, it remains mostly unclear how R248W mutation induces destabilization of p53C and how ADH-6 stabilizes this p53C mutant and inhibits its aggregation. Herein, we conducted all-atom molecular dynamics simulations of R248W p53C in the absence and presence of ADH-6, as well as that of wild-type (WT) p53C. Our simulations reveal that the R248W mutation results in a shift of helix H2 and ß-hairpin S2-S2' towards the mutation site, leading to the destruction of their neighboring ß-sheet structure. This further facilitates the formation of a cavity in the hydrophobic core, and reduces the stability of the ß-sandwich. Importantly, two crucial aggregation-prone regions (APRs) S9 and S10 are disturbed and more exposed to solvent in R248W p53C, which is conducive to p53C aggregation. Intriguingly, ADH-6 dynamically binds to the mutation site and multiple destabilized regions in R248W p53C, partially inhibiting the shift of helix H2 and ß-hairpin S2-S2', thus preventing the disruption of the ß-sheets and the formation of the cavity. ADH-6 also reduces the solvent exposure of APRs S9 and S10, which disfavors the aggregation of R248W p53C. Moreover, ADH-6 can preserve the WT-like dynamical network of R248W p53C. Our study elucidates the mechanisms underlying the oncogenic R248W mutation induced p53C destabilization and the structural protection of p53C by ADH-6.

Structural diversity in the membrane-bound hIAPP dimer correlated with distinct membrane disruption mechanisms.

Amyloid deposits of the human islet amyloid polypeptide (hIAPP) have been identified in 90% of patients with type II diabetes. Cellular membranes accelerate the hIAPP fibrillation, and the integrity of membranes is also disrupted at the same time, leading to the apoptosis of ß cells in pancreas. The molecular mechanism of hIAPP-induced membrane disruption, especially during the initial membrane disruption stage, has not been well understood yet. Herein, we carried out extensive all-atom molecular dynamics simulations investigating the hIAPP dimerization process in the anionic POPG membrane, to provide the detailed molecular mechanisms during the initial hIAPP aggregation stage in the membrane environment. Compared to the hIAPP monomer on the membrane, we observed not only an increase of α-helical structures, but also a substantial increase of ß-sheet structures upon spontaneous dimerization. Moreover, the random coiled and α-helical dimer structures insert deep into the membrane interior with a few inter-chain contacts at the C-terminal region, while the ß-sheet-rich structures reside on the membrane surface accompanied by strong inter-chain hydrophobic interactions. The coexistence of α and ß structures constitutes a diverse structural ensemble of the membrane-bound hIAPP dimer. From α-helical to ß-sheet structures, the degree of membrane disruption decreases gradually, and thus the membrane damage induced by random coiled and α-helical structures precedes that induced by ß-sheet structures. We speculate that insertion of random coiled and α-helical structures contributes to the initial stage of membrane damage, while ß-sheet structures on the membrane surface are more involved in the later stage of fibril-induced membrane disruption.

Elucidating the reversible and irreversible self-assembly mechanisms of low-complexity aromatic-rich kinked peptides and steric zipper peptides.

Many RNA-binding proteins such as fused-in sarcoma (FUS) can self-assemble into reversible liquid droplets and fibrils through the self-association of their low-complexity (LC) domains. Recent experiments have revealed that SYG-rich segments in the FUS LC domains play critical roles in the reversible self-assembly behaviors of FUS. These FUS LC segments alone can self-assemble into reversible kinked fibrils, which are markedly different from the canonical irreversible steric zipper ß-sheet fibrils. However, the molecular determinants underlying the reversible and irreversible self-assembly are poorly understood. Herein we conducted extensive all-atom and coarse-grained molecular dynamics simulations of four representative hexapeptides: two low-complexity aromatic-rich kinked peptides from the amyotrophic lateral sclerosis-related FUS protein, FUS37-42 (SYSGYS) and FUS54-59 (SYSSYG); and two steric zipper peptides from Alzheimer's-associated Aß and Tau proteins, Aß16-21 (KLVFFA) and Tau306-311 (VQIVYK). We dissected their reversible and irreversible self-assembly dynamics, predicted their phase separation behaviors, and elucidated the underpinning molecular interactions. Our simulations showed that alternating stickers (Tyr) and spacers (Gly and Ser) in FUS37-42 and FUS54-59 facilitate the formation of highly dynamic coil-rich oligomers and lead to reversible self-assembly, while consecutive hydrophobic residues of LVFF in Aß16-21 and IVY in Tau306-311 act as hydrophobic patches, favoring the formation of stable ß-sheet-rich oligomers and driving the irreversible self-assembly. Intriguingly, we found that FUS37-42 and FUS54-59 peptides, possessing the same amino acid composition and the same number of sticker and spacer residues, display differential self-assembly propensities. This finding suggests that the self-assembly behaviors of FUS peptides are fine-tuned by the site-specific patterning of spacer residues (Ser and Gly). This study provides significant mechanistic insights into reversible and irreversible peptide self-assembly, which would be helpful for understanding the molecular mechanisms underlying the formation of biological liquid condensates and pathological solid amyloid fibrils.