Integrating smFRET, SAXS and NMR data to infer structural ensembles of an intrinsically-disordered protein

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging. Transient long-range interactions in IDPs are known to have important functional implications. Thus, in order to calculate reliable structural ensembles of IDPs, the data used in their calculation must capture these important structural features. We use integrative modelling to understand and implement conformational restraints imposed by the most common structural techniques for IDPs: NMR spectroscopy, small-angle X-ray scattering (SAXS), and single-molecule Forster Resonance Energy Transfer (smFRET). Using the disordered N-terminal region of the Sic1 protein as a test case, we find that only Paramagnetic Relaxation Enhancement (PRE) and smFRET measurements are able to unambiguously report on transient long-range interactions. It is precisely these features which lead to deviations from homopolymer statistics and divergent structural inferences in non-integrative smFRET and SAXS analysis. Furthermore, we find that the sequence-specific deviations from homopolymer statistics are consistent with biophysical models of Sic1 function that are mediated by phospho-sensitive binding to its partner Cdc4. To our knowledge, these are the first conformational ensembles for an IDP in physiological conditions that are simultaneously consistent with smFRET, SAXS, and NMR data. Our results stress the importance of integrating the global and local structural information provided by SAXS and Chemical Shifts, respectively, with information on specific inter-residue distances from PRE and smFRET. Our integrative modelling approach and quantitative polymer-physics-based characterization of the experimentally-restrained ensembles could be used to implement a rigorous taxonomy for the description and classification of IDPs as heteropolymers.