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bioRxiv · 10.64898/2026.01.08.698362

CD2AP's structure and oligomerization are compromised by the K301M mutation: implications for Nephrotic syndrome

Qadri, A. H.·Trapathi, R. M.·Faheem, I.·Meshram, S.·Reddem,, E. R.·Ramachandran, R.·Pasupulati, A. k.
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Abstract

IntroductionThe podocyte slit diaphragm (SD) is a complex filtration unit localized to the blood and urine interface and governs the glomerular selectivity. However, the greater details of the SD composition and the mechanism of assembly of the SD protein as a macromolecular complex remain elusive. CD2-associated protein (CD2AP) serves as a central scaffold within the SD, and mutations in CD2AP are strongly associated with nephrotic syndrome (NS) and focal segmental glomerulosclerosis (FSGS). However, the mechanisms by which such mutations alter the architecture and higher-order organization of CD2AP are poorly understood. MethodsWe employed biophysical, structural, and proteomic approaches to investigate the impact of the disease-associated K301M mutation on CD2AP structure and its interaction with Podocin. Oligomerization was analyzed using size-exclusion chromatography, blue native PAGE, Dynamic light scattering, and small-angle X-ray scattering. Secondary and tertiary structural properties were assessed by far- and near-UV circular dichroism, thermal denaturation, and intrinsic fluorescence spectroscopy. CD2AP-podocin interactions were quantified using in vitro pulldown and surface plasmon resonance (SPR), and mutation-dependent changes in interaction networks were examined through interactome profiling. ResultsWild-type (WT) CD2AP assembled into flexible higher-order oligomers ([~]9-12-mers), whereas the K301M variant collapsed into lower-order species ([~]3-6-mers), indicating destabilization of the coiled-coil assembly interface. Spectroscopic analyses revealed subtle secondary-structure rearrangements, but profound tertiary packing defects, as well as reduced and markedly diminished thermal resilience in the mutant. SPR analysis demonstrated loss of binding between Podocin and mutant CD2AP, whereas WT CD2AP showed high-affinity interaction (KD = 211 nM) with Podocin. Complementary interactome profiling revealed widespread rewiring of protein-protein interactions in the case of mutant CD2AP, characterized by the loss of core partners and the emergence of aberrant associations. ConclusionThese findings define a mechanistic model in which the K301M mutation destabilizes CD2AP oligomerization, disrupts podocin recognition, and remodels interaction networks essential for SD stability. This work signifies the importance of CD2AP in SD assembly and the permselective filtration function of the kidney, and the impact of a single mutation in the pathogenesis of NS and FSGS. Translational Statement: Inherited nephrotic syndrome, characterized by heavy proteinuria, frequently arises from mutations in scaffolding proteins of the slit-diaphragm (SD). This study demonstrates that the nephrotic syndrome-associated K301M mutation in CD2-associated protein (CD2AP) compromises higher-order oligomerization, abolishes its binding to the binding partner (Podocin), and reshapes protein-protein interaction networks that are critical for SD assembly and stability. By establishing a direct link between mutation-induced collapse of CD2AP architecture and loss of podocyte scaffolding function, these findings provide mechanistic insight into the pathogenesis of CD2AP-associated proteinuric kidney disease. The results further identify oligomeric assembly interfaces as potential targets for therapeutic strategies aimed at preserving the integrity of the SD and glomerular filtration function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/698362v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1f49f1dorg.highwire.dtl.DTLVardef@fa2e06org.highwire.dtl.DTLVardef@e399c0org.highwire.dtl.DTLVardef@831c17_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

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Qadri, A. H., Trapathi, R. M., Faheem, I., Meshram, S., Reddem,, E. R., Ramachandran, R., Pasupulati, A. k.. 2026-01-09. CD2AP's structure and oligomerization are compromised by the K301M mutation: implications for Nephrotic syndrome. https://doi.org/10.64898/2026.01.08.698362

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