The role of Wnt palmitoleylated loop conserved disulfide bonds in Wnt-frizzled complex structural dynamics: Insights from molecular dynamics simulations

Wnts are lipid-modified proteins rich in cysteine, regulating developmental processes, and are involved in various pathological conditions. Wnts structure resembles a hand, with a palmitoleylated thumb and an index finger-like domain interacting with frizzled (FZ) receptors. Previous research shows the palmitoleyl group and the disulfides importance in Wnt folding, secretion, and function, but the structural basis is not fully understood. Here, we utilized classical molecular dynamics simulation (800-ns in total) to investigate how the thumb palmitoleyl and its close conserved disulfides (183-190, 181-195) regulated Wnt-FZ interaction and structural dynamics. Using Steered molecular dynamics experiment followed by a relaxing procedure, we also explored if these disulfides are important in Wnt-FZ complex formation. According to our results, the palmitoleyl group contributes significantly to stabilize Wnt-FZ interaction, and the disulfides modulate this contribution. We also demonstrated that disulfide 183-190 regulates the Wnt thumb fluctuation, hydrogen bond network, and secondary structure. The DCCM analysis depicted disulfide 183-190 roles in regulating native-like collective movement in the palmitoleylated loop, which changed after this disulfide removal. The pulling-relaxing experiment showed that both the disulfides, and especially, the disulfide 183-190, are highly important for long-range salt-bridge interaction establishment between Wnt Lys182 and FZ Glu64, led palmitoleyl group appropriate positioning to FZ, suggested this disulfide essential role in Wnt-FZ complex formation. Together, our findings provide new insights to how thumb-positioned disulfides contribute to Wnt-FZ complex formation, structural dynamics, and stability, introducing disulfide 183-190 as a consequential element to target in drug design and development against Wnt signalling.