060 | Interdomain interactions and pH modulate folding intermediates in MAGI1 PDZ4

Cosmin Marian Obreja¹, Valeria Pennacchietti², Dimitrios Marinidis¹, Sara Di Matteo³, Rodolfo Ippoliti¹, Stefano Gianni², Francesca Malagrinò¹ | ¹University of L’Aquila, Italy; ²University “La Sapienza” of Rome, Italy; ³University of Chieti-Pescara, Italy.

Membrane-associated guanylate kinases (MAGUKs) are scaffold proteins that orchestrate signaling assemblies and preserve junctional integrity through modular interaction domains, prominently PDZ domains [1]. The multi-PDZ scaffold MAGI1 is a key organizer at cell–cell junctions and its dysregulation has been linked to tumor progression [2], motivating a mechanistic understanding of how its modular architecture shapes domain behavior. PDZ domains are also well-established models to interrogate protein folding principles, as they share conserved folds and can populate transient intermediates [3]. Here, we combined equilibrium denaturation and kinetic (un)folding measurements to dissect the folding landscape of the PDZ4 domain of MAGI1, both in isolation and within multidomain constructs that mimic its native context (PDZ3–4 and PDZ2–6) [4]. PDZ4 alone deviates from a two-state mechanism, displaying a rollover effect consistent with the accumulation of a refolding intermediate. In addition, this kinetic signature is markedly enhanced in multidomain contexts, indicating that adjacent domains influence the folding pathway without substantially altering overall thermodynamic stability. Global kinetic analysis supports the population of a distinct intermediate in PDZ3–4 and PDZ2–6 that is stabilized by non-native contacts, pointing to cooperative, context-dependent interactions across domain boundaries. We further probed environmental regulation by varying pH: acidic conditions destabilize the native state of isolated PDZ4, consistent with disruption of key electrostatic interactions, yet selectively promote stabilization of the intermediate only in multidomain constructs. Structural inspection suggests that specific salt bridges may contribute to this pH-dependent stabilization of the misfolded species. Overall, our results indicate that misfolding in MAGI1 PDZ4 is not simply a passive consequence of folding, but an emergent feature of multidomain organization that can be tuned by solution conditions. This work expands our understanding of protein (mis)folding in multidomain systems and could provide mechanistic insight into how scaffold proteins can encode folding plasticity through their modular organization.