Suchergebnisse

Many intrinsically disordered proteins (IDPs) may undergo liquid-liquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalization of intracellular biochemical reactions. The phase behavior of IDPs is sequence dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intramolecular and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS. ; We thank Veronica Ryan and Nicolas L. Fawzi for sharing the PRE data for FUS LCD, FUS12E LCD, and A2 LCD as well as Robert Konrat for sharing the intramolecular PRE data for Osteopontin. We thank Robert B. Best for sharing data on compaction of IDPs; Gregory L. Dignon and Jeetain Mittal for help setting up simulations with the HPS model; and Tanja Mittag, Massimiliano Bonomi, and Benjamin Schuler for helpful discussions. We acknowledge funding from the BRAINSTRUC structural biology initiative from the Lundbeck Foundation (Grant R155-2015-2666) and acknowledge access to computational resources from the Resource for Biomolecular Simulations (ROBUST) (supported by Novo Nordisk Foundation Grant NNF18OC0032608) and Biocomputing Core Facility at the Department of Biology, University of Copenhagen. T.K.S. acknowledges support from the Novo Scholarship Programme 2021. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement 101025063. ; Peer reviewed

p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF-PCNA-DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η-PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass. ; Italian Association for Cancer Research [iCARE fellowship from AIRC and the European Commission to A.D.B. and AIRC Grant IG14718 to S.O., IG20762 to G.M and MFAG18811 to E.C.]; Spanish Ministry of Economy and Competitiveness [CTQ2017-83810-R grant to F.J.B.]; CIC bioGUNE acknowledges MINECO for the Severo Ochoa Excellence Accreditation [SEV-2016-0644]; S.B-V. and A.G.-M. acknowledge fellowships from MINECO [BES-2013-063991 and BES-2015-075847]; M.R.-M. is supported by a pre-doctoral fellowship from the Basque Government [PRE_2016_2_0249]. Funding for open access charge: University of Leicester. ; Peer reviewed