title: Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study.
abstract: Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.
authors.affiliation: University Grenoble Alpes, ‡CNRS, and §CEA, Protein Dynamics and Flexibility, Institut de Biologie Structurale , 38000 Grenoble, France.
authors.forename: Jie-rong
authors.initials: JR
authors.lastname: Huang
authors.forename: Lisa R
authors.initials: LR
authors.lastname: Warner
authors.forename: Carolina
authors.initials: C
authors.lastname: Sanchez
authors.forename: Frank
authors.initials: F
authors.lastname: Gabel
authors.forename: Tobias
authors.initials: T
authors.lastname: Madl
authors.forename: Cameron D
authors.initials: CD
authors.lastname: Mackereth
authors.forename: Michael
authors.initials: M
authors.lastname: Sattler
authors.forename: Martin
authors.initials: M
authors.lastname: Blackledge
mesh_terms: D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D053838:Scattering, Small Angle; D000072668:Splicing Factor U2AF; D055672:Static Electricity; D014961:X-Ray Diffraction
chemical_list: D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D012313:RNA