title: Theoretical modeling of multiprotein complexes by iSPOT: Integration of small-angle X-ray scattering, hydroxyl radical footprinting, and computational docking.
abstract: Structural determination of protein-protein complexes such as multidomain nuclear receptors has been challenging for high-resolution structural techniques. Here, we present a combined use of multiple biophysical methods, termed iSPOT, an integration of shape information from small-angle X-ray scattering (SAXS), protection factors probed by hydroxyl radical footprinting, and a large series of computationally docked conformations from rigid-body or molecular dynamics (MD) simulations. Specifically tested on two model systems, the power of iSPOT is demonstrated to accurately predict the structures of a large protein-protein complex (TGFβ-FKBP12) and a multidomain nuclear receptor homodimer (HNF-4α), based on the structures of individual components of the complexes. Although neither SAXS nor footprinting alone can yield an unambiguous picture for each complex, the combination of both, seamlessly integrated in iSPOT, narrows down the best-fit structures that are about 3.2Å and 4.2Å in RMSD from their corresponding crystal structures, respectively. Furthermore, this proof-of-principle study based on the data synthetically derived from available crystal structures shows that the iSPOT-using either rigid-body or MD-based flexible docking-is capable of overcoming the shortcomings of standalone computational methods, especially for HNF-4α. By taking advantage of the integration of SAXS-based shape information and footprinting-based protection/accessibility as well as computational docking, this iSPOT platform is set to be a powerful approach towards accurate integrated modeling of many challenging multiprotein complexes.
authors: Center for Proteomics and Department of Nutrition, Case Western Reserve University, Cleveland, OH, USA.
authors: Wei
authors: W
authors: Huang
authors: Center for Proteomics and Department of Nutrition, Case Western Reserve University, Cleveland, OH, USA.
authors: Krishnakumar M
authors: KM
authors: Ravikumar
authors: Alan Edwards Centre for Research on Pain, McGill University, Montreal, Quebec, Canada.
authors: Marc
authors: M
authors: Parisien
authors: Center for Proteomics and Department of Nutrition, Case Western Reserve University, Cleveland, OH, USA. Electronic address: sichun.yang@case.edu.
authors: Sichun
authors: S
authors: Yang
mesh_terms: D055592:Biophysical Phenomena; D051557:Hepatocyte Nuclear Factor 4 / Q000737:chemistry*; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D046912:Multiprotein Complexes / Q000737:chemistry / Q000648:ultrastructure; D011487:Protein Conformation; D053838:Scattering, Small Angle; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry*; D016212:Transforming Growth Factor beta / Q000737:chemistry*; D014961:X-Ray Diffraction; D014965:X-Rays
chemical_list: C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D046912:Multiprotein Complexes; D016212:Transforming Growth Factor beta; D017665:Hydroxyl Radical; D022061:Tacrolimus Binding Protein 1A
keywords: Footprinting; Integration; Modeling; SAXS