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Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils

David S. Yang,A. Saeedi,Mohsen Fathi,M. Sherman,M. Safari,Alena Klindziuk,M. Barton,N. Varadarajan,A. Kolomeisky,P. Vekilov

Published 2021 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance The mesoscopic p53-rich clusters that we discover represent a new class of biological condensate, distinct from amorphous and ordered aggregates and the dense liquids found with several physiologically active proteins. The demonstrated two-step mechanism of amyloid fibril nucleation, whereby the clusters host the nucleation of p53 amyloid fibrils, illustrates the potency of recently identified nonclassical nucleation concepts to understand intracellular processes. This finding establishes a new biophysical paradigm for the assembly of numerous ordered functional and pathological biological solids, such as tubules, filaments, sickle cell polymers, amyloids, and crystals. The protein p53 is a crucial tumor suppressor, often called “the guardian of the genome”; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

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