Structural conformations of intrinsically disordered proteins of podocyte slit-diaphragm

The slit-diaphragm (SD), a specialized junction between the foot processes of neighboring podocytes, regulates the permselectivity of glomerular filtration. SD functions as a size- and charge-selective molecular sieve, accounting for the formation of ultrafiltered protein-free urine. SD comprises several proteins such as podocin, CD2AP, NEPH1, TRPC6, and nephrin. The extracellular region of Nephrin and NEPH1 bridges the gap between foot processes. Podocin and CD2AP serve as adapter proteins. The intrinsically disordered regions (IDRs) of SD proteins play a crucial role in assembling individual proteins into a macromolecular complex. Mutations in SD proteins cause nephrotic syndrome (NS), characterized by heavy proteinuria. The mechanistic insights into how SD proteins assemble as macromolecular complexes remained unknown. Further, how mutations distort the interactions among SD proteins remains elusive. We employed docking and molecular dynamics simulations to investigate the structural dynamics of SD proteins. Our findings reveal that SD proteins exhibit partner-specific adaptations. Short linear motifs and molecular recognition features drive these adaptations. CD2AP interacts transiently with nephrin but forms a more stable complex with podocin. NEPH1 and nephrin interact via their extracellular immunoglobulin domains. Podocin shows preferential interactions with nephrin over NEPH1. Distinct subunit-sharing mechanisms emerge in which CD2AP and TRPC6 can simultaneously bind to a single podocin subunit. Mutational analysis reveals that disease-associated variants (CD2AP (P532S), podocin (R138Q), and nephrin (G1161V)) enhance local stability but restrict the flexibility of IDRs and impair the SD assembly. Our study provides evidence of dynamic subunit sharing within the SD complex and a mechanistic basis for NS. Interacting Interfaces of Target SD Proteins A predictive assembly of slit-diaphragm proteins (AlphaFold models in sphere representation) highlighting key protein interactions in the podocyte slit-diaphragm. The proteins include Podocin (N-terminal: brown, C-terminal: red), CD2AP (SH3 domains: hot pink, C-terminal: magenta), NEPH1 (extracellular: green), TRPC6 (purple), and nephrin (extracellular: blue). Transmembrane regions are shown in grey and present in all proteins except CD2AP. Podocin interacts with CD2AP, the intracellular domains of NEPH1 and nephrin, and the C-terminal of TRPC6. CD2AP engages with the C-terminal domains of podocin and nephrin. NEPH1 connects extracellularly and intracellularly with nephrin and interacts with podocin's C-terminal domain. TRPC6 interfaces with nephrin's intracellular region through its N-terminal. Note that not all potential interactions are depicted.