Covalent linkage of the papillomavirus E2 DNA-binding domain reveals a soluble amyloid-like assembly pathway

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Abstract Early soluble intermediates in amyloid formation are difficult to characterize because they are transient and obscured by competing insoluble aggregation. Here, we use scE2C, an engineered covalently linked version of the HPV16 E2 DNA-binding dimer, as a tractable model to stabilize and characterize soluble amyloid-like assemblies that are poorly resolved in the native dimeric protein. Whereas native E2C progresses toward visible insoluble aggregation upon heating, scE2C undergoes an irreversible transition to a soluble β-rich state. This state displays amyloid-sensitive signatures, including enhanced thioflavin T fluorescence, a Congo Red spectral shift, and increased ANS binding. Dynamic light scattering and atomic force microscopy revealed soluble assemblies comprising small spherical particles, larger spherical particles, and worm-like protofibrillar structures, which were further visualized by negative-stain and cryo-electron microscopy. Quantitative comparison of DLS- and AFM-derived dimensions supports a hierarchical organization in which smaller spherical particles behave as building blocks for larger particles and necklace-like worms. Isothermal circular dichroism kinetics across concentration and temperature were consistent with an assembly-coupled structural transition, with an apparent low-order nucleus of approximately 2 ± 1 scE2C protomeric units and growth compatible with low-order addition. Specific DNA binding fully inhibited the thermal transition, indicating that access to this protofibrillar branch is constrained by the functional DNA-bound state. Together, these results support the view that covalent linkage of the E2C dimer can provide experimental access to soluble early amyloid-like assembly states under conditions where native E2C progresses toward insoluble aggregation.

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