Stability and interaction defects in the Sin3A-PAH1 αα-hub domain associated with loss-of-function variants

Hub proteins organize large interactomes using their hub domains for protein-protein interactions. αα-hub domains are present in transcriptional regulators, such as Switch-insensitive 3A (Sin3A), an integral scaffolding protein of histone deacetylation complexes. Here, we relate αα-hub stability to function by structural and thermodynamic studies of the Sin3A-paired amphipathic helix 1 (PAH1) wt hub domain and two predicted loss-of-function variants (A126V and K155E) found in patients with the neurodevelopmental Witteveen-Kolk syndrome. For an αα-hub domain, the PAH1 domain is relatively stable with a ΔG_DN of 14.1 ± 0.9 kJ mol^-1, which likely compromises its adaptability in binding, thereby increasing specificity. For the two known binding partners, ten-eleven translocase 1 (Tet1) and Sin3A-associated protein 25 (SAP25), a sequence motif undergoes coupled folding and binding with Sin3A-PAH1. The SAP25 interaction shows prototypical thermodynamics for a disorder-based interaction with an enthalpy-driven interaction counteracted by an entropic penalty. In contrast, for the Tet1 interaction, there is no net contribution from entropy to binding, even when including a disordered context in the motif-containing Tet1 fragment. The variants of Sin3A-PAH1 exhibit lower affinities for both SAP25 and Tet1, with a >15-fold reduction in the affinity of PAH1-K155E for Tet1. Notably, the A126V side-chain substitution results in an increased global stability (ΔΔG_DN = 6.9 kJ mol^-1), representing a case of increased stability associated with loss of function. Our findings show how disease-associated Sin3A-PAH1 variants can affect global domain stability and flexibility as well as interactions with specific partner proteins, thereby contributing to the deconvolution of signal fidelity governed by folded hubs in interactions with disordered partner proteins.