Dissecting the structural features of folding asymmetry and transient misfolding in a multidomain PDZ scaffold

Understanding how multidomain proteins fold, avoid misfolding, and maintain functional regulation represents a critical problem in structural biology, with broad relevance for biotechnology. In this study, the tandem PDZ1–PDZ2 domains of the scaffold protein X11, a system of particular interest due to its previously reported asymmetric folding and unfolding behavior as well as its propensity to form transient misfolded intermediates, are investigated. Through extensive mutational work and in-depth kinetic folding analysis, the folding behavior of this multidomain construct is dissected, and a comparative analysis with its isolated PDZ domains is also performed. The results reveal that folding and unfolding proceed through distinct pathways with PDZ2 folding rapidly and independently, while PDZ1 folds more slowly and only upon engagement of an autoinhibitory regulatory tail. Despite these differences, the folding mechanisms of each domain are conserved when studied in isolation, with deviations largely confined to functionally relevant and frustrated regions. The results also allow to depict the structural features of a misfolded intermediate that competes with productive folding and is stabilized by non-native interdomain contacts. Strikingly, this misfolded trap retains elements of the PDZ2 folding nucleus, an unexpected finding that allows us to draw broader conclusions about how transient misfolding can arise even from native-like structural motifs. We discuss these results in light of prior studies on multidomain proteins.