Podocytes are crucial cells of the glomerular filtration unit and plays a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and charge-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101–125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126–350). We show that the podocin domain majorly homo-oligomerizes into a 16-mer. Circular dichroism and fluorescence spectroscopy suggest that the 16-mer oligomer has considerable secondary structure and moderate tertiary packing.

Podocytes are crucial cells of the glomerular filtration unit and playing a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and shape-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101-125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126-350). We show that the podocin domain majorly homo-oligomerize into a 16mer. Circular dichroism and fluorescence spectroscopy suggest that the 16mer oligomer has considerable secondary structure and moderate tertiary packing.

Single-stranded DNA-binding protein (SSB) coats single-stranded DNA (ssDNA) and recruits downstream factors, yet this same coating must be cleared for SSB-interacting proteins to bind. RecA is a central protein in homologous recombination, which requires SSB-free ssDNA to load during the process. RecO, a recombination mediator protein, is thought to regulate this handoff, but it has not been shown directly that RecO drives the release of full-length, ssDNA-bound SSB. Using atomic force microscopy, we show that RecO actively displaces full-length wild-type SSB from a defined ssDNA substrate, reducing occupancy from 67% to 29%. Displacement efficiency decreases after deleting the C-terminal tip of SSB (ΔC8) or after adding a single alanine at the C-terminus of SSB (A179). Molecular dynamics simulations show that A179 retains its native RecO contact but additionally engages a non-native surface, producing a specific, over-engaged complex, suggesting that these interprotein interactions may affect SSB-ssDNA contacts. Together, these findings suggest that RecO-driven SSB clearance is governed by the protein–protein interface rather than by ssDNA-binding affinity alone, thereby acting as a checkpoint against indiscriminate SSB removal during genome maintenance.

Single-stranded DNA-binding protein (SSB) coats single-stranded DNA (ssDNA) and recruits downstream repair factors through its acidic C-terminal tip, yet this same coating must be cleared for RecA – a central protein in homologous recombination, to load during the process. RecO, a recombination mediator protein, is thought to regulate this handoff, but whether and how it drives release of full-length, ssDNA-bound SSB, rather than simply docking onto an isolated SSB peptide as captured in the only available crystal structure, has not been shown directly. Using single-molecule atomic force microscopy, we show that RecO actively displaces full-length wild-type SSB from a defined ssDNA substrate, reducing occupancy from 67% to 29%. Displacement efficiency decreases after deleting the SSB C-terminal tip (ΔC8) or extending it by a single residue (A179); the latter instead increases ssDNA occupancy and complex volume. Molecular dynamics simula-tions show that A179 retains its native RecO contact but additionally engages a non-native surface, producing a specific, over-engaged complex rather than non-specific aggregation suggesting that these interprotein interactions can affect SSB-ssDNA contacts. Together, these results suggest that RecO-driven SSB clearance is governed by the precise geometry of a single protein-protein interface rather than by affinity alone: too little engagement (ΔC8) or too much (A179) both abolish displacement, acting as a checkpoint against in-discriminate SSB removal during genome maintenance.