Methodological development in BioDisOrder has been focused so far on the employment of artificial intelligence (Qi et al J Chem Theory Comput. 2022 18:7733-7750) to analyse nuclear magnetic resonance (NMR) data (Gallo et al, JACS AU 2024 4:2372-2380) for characterising the disordered regions of membrane proteins. This has enabled the definition of new approaches to refine the structural ensembles of protein molecules and membranes with significant accuracy, including structure and dynamics of disordered protein regions that are impossible to characterise with current methods. The method included the development of coarse grained MD potentials to enhance the conformational search of disordered regions under the guidance of NMR data (Navarro Paya et al, Front Mol Biosci. 2022 9:857217).
We have applied these methodological innovations to different topics in the context of disordered regions of membrane-associated proteins. In particular, we have showed that the disordered binding of alpha-synuclein to the internal plasma membrane enables to stabilise the docking of synaptic vesicles in such a way to contribute to their exocytosis process (Man et al, Nature Communications 2021, 12:927). In the context of aberrant aggregation of alpha-synuclein, the central protein in Parkinson's disease, we have used the methods of BioDisOrder to achieve an unprecedented characterisation of the structure of transient fibrils from liquid-liquid phase separations (Chen et al, JACS 2024, 146:10537-10549). Additional studies on alpha-synuclein involved its N-terminal region and its role in membrane binding (Runfola et al, Sci Rep. 2020 10(1):204) or aggregation (Stephens et al, Nat Commun. 2020 11:2820). The interdisciplinary approach also enabled to study the conformational transitions leading to the aggregation of the Prion Protein and its pathological mutant T183A (Sanz-Hernandez et al, PNAS 2021, 118: e2019631118) as well as put the basis for a new drug discovery platform against Parkinson's disease (Palmas et al Neurotherapeutics. 2022 19:305-324). Another successful application of BioDisOrder has enabled a unique structural refinement of the membrane-protein complex between SERCA and PLN (Weber et al eLife 2021, 10:e66226), a key complex for the regulation of the heart cycles. This work was carried out in collaboration with the Veglia lab at the University of Minnesota. The structure of this fundamental complex for the heart function and pathology has represented a top challenge for decades. Previous advancements in this research area enabled to crystallise the SERCA-PLN complex, however, the resulting structure didn't reveal the key N-terminal regulatory domain PLN, which is disordered in the complex. Using the methods developed in BioDisOrder, we obtained the first atomic-resolution ensemble of the complex, including the disordered N-terminal region of PLN that regulate SERCA. The work also clarified how the phosphorylation of Ser 16 of PLN enhances the activity of the SERCA calcium pump in the complex. Other applications have included the study of the dynamics of thermophilic protein at high temperatures using the combination of NMR and theory (Fusco et al Front Mol Biosci. 2022 9:981312) or the role of cholesterol in influencing the binding of disordered proteins (Man et al Front Neurosci. 2020 14:18).
