Solution Structure Analysis of Cytoplasmic Domain of Podocyte Protein Neph1 Using Small/Wide Angle X-ray

Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of the Neph1 cytoplasmic domain (Neph1-CD) is required.Inthisstudy,thesolutionstructureofthisdomainwas determined by small/wide angle x-ray scattering (SWAXS). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 A u, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The HisZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD,andthecrystalstructureofZO-1-PDZ1toconstruct a structural model of the Neph1-CD zZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues Thr-His-Val, residues Lys761 and Tyr-762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and invivo andinvitro pull-downexperimentsdemonstratedlossof binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that providesnovelinsightsintothemolecularstructureandfunctionof Neph1-CD.