LAUSR

Ubiquitin-modified proliferating cell nuclear antigen (PCNA) and small ubiquitin-like modifier (SUMO)-modified PCNA regulate DNA damage tolerance pathways. X-ray crystal structures of these proteins suggested that they do not have much conformational flexibility because the modifiers have preferred binding sites on the surface of PCNA. By contrast, small-angle X-ray scattering analyses of these proteins suggested that they have different degrees of conformational flexibility, with SUMO-modified PCNA being more flexible. These conclusions were based on minimal-ensemble hybrid approaches, which produce unrealistic models by representing flexible proteins with only a few static structures. To overcome the limitations of minimal-ensemble hybrid approaches and to determine the degree of conformational flexibility of ubiquitin-modified PCNA and SUMO-modified PCNA, we utilized a novel full-ensemble hybrid approach. We carried out molecular simulations and small-angle X-ray scattering analyses of both proteins and obtained outstanding agreement between the full ensembles generated by the simulations and the experimental data. We found that both proteins have a high degree of conformational flexibility. The modifiers occupy many positions around the back and side of the PCNA ring. Moreover, we found no preferred ubiquitin-binding or SUMO-binding sites on PCNA. This conformational flexibility likely facilitates the recognition of downstream effector proteins and the formation of PCNA tool belts.