Adjacent domains drive the emergence of misfolded intermediates in the folding pathway of the PDZ4 from MAGI1.

Protein misfolding in multidomain assemblies emerges from a complex interplay between intra- and interdomain interactions. Here, we dissect how neighboring domains shape the folding and misfolding of the PDZ4 domain from the scaffold protein MAGI1. Exploiting the single intrinsic tryptophan in PDZ4, we monitored its behavior in the isolated domain, in the PDZ3-4 tandem, and within the five-domain PDZ2-6 supramodule by equilibrium and kinetic (un)folding experiments over a broad pH range. Equilibrium denaturation reveals that PDZ4 displays a cooperative, two-state-like transition with similar thermodynamic stability in all constructs, indicating that domain adjacency does not appreciably affect its native state. In contrast, kinetic measurements uncover pronounced deviations from two-state behavior. PDZ4 alone populates a transient intermediate, whose population and associated kinetic slowdown are markedly amplified in the tandem and even more in the supramodular context, consistent with the formation of a misfolded ensembles stabilized by non-native interdomain contacts. Acidic pH selectively enhances the kinetic trap in PDZ3-4 and PDZ2-6, but not in PDZ4 alone, pointing to a key role of electrostatics at interdomain interfaces, potentially involving specific salt bridges. Structural inspection suggests the presence of potential specific salt bridges (Asp871/Glu901 with Arg873/Lys905), although further investigation is required. Our data show that misfolding of MAGI1 PDZ4 is a context-dependent, cooperative property of adjacent domains rather than a simple by-product of folding, and illustrate how supramodular organization encodes kinetic plasticity in scaffold proteins.