Scaffold proteins frequently employ tandem interaction domains to achieve affinity and specificity beyond that of individual modules. The MAGI-1 scaffold, a member of the membrane-associated guanylate kinase family, contains a central WW tandem whose mechanistic contribution to ligand recognition has remained unclear. Here, we dissect the binding properties of the MAGI-1 WW tandem using time-resolved kinetic analyses, complemented by calorimetric measurements, and show that the tandem architecture substantially enhances affinity towards bidentate ligands by stabilizing the bound complex. Furthermore, we demonstrate binding to proceed through a conformational selection mechanism, whereby the tandem samples alternative conformational states before high-affinity engagement. This response is further modulated by environmental pH, with cooperative dissociation transitions linked to electrostatic contacts within the tandem. Importantly, ligand recognition is highly sensitive to inter-motif spacing; swapping naturally occurring long and short linkers between PY alter complex stability, with longer linkers weaking binding. These findings suggest that the MAGI-1 WW tandem acts as a dynamic supradomain, thereby escaping the promiscuity, classically observed in single WW modules, by integrating geometric and conformational signals which enable a selective regulation of protein-protein interaction networks.
Toso et al. (2026) studied this question.