Structural and functional effects of carbonylation and phosphorylation on the Zu5-ANK domain: A molecular dynamics and docking study.

4-HNE carbonylation and phosphorylation in the Zu5-ANK domain have been observed in human erythrocytes with sickle cell trait and G6PD deficiency. However, the atomic-level effects of phosphorylation and multiple carbonylations by 4-HNE on the Zu5-ANK domain are not yet fully understood. The present work provides further insight into understanding the changes caused by polymodifications in Zu5-ANK due to oxidative stress and phosphorylation. Here, we present a molecular dynamics simulation-based study of the polycarbonylated, phosphorylated, and phosphocarbonylated Zu5-ANK domain. Our results show that these modifications induce local conformational and mobility changes in the protein, particularly within three regions, which include specific residues that interact with the β-spectrin protein. Furthermore, our analyses revealed that while the domain exhibits remarkable global structural resilience to isolated modifications, the combination of phosphorylation and polycarbonylation exerts a synergistic destabilizing effect. This polymodified state alters the spatial geometry of the basic residue patch essential for β-spectrin binding. Subsequent protein-protein docking with β-spectrin repeats 14 and 15 demonstrated that although isolated carbonylation is structurally tolerated at the interface, the simultaneous presence of both modifications disrupts the canonical electrostatic complementarity. This perturbation leads to a reduced buried surface area, diminished interaction specificity, and a highly heterogeneous binding mode. Overall, these in silico findings propose a theoretical molecular rationale for how the accumulation of multiple post-translational modifications might weaken the ankyrin-spectrin anchorage, providing a mechanistic basis for future experimental studies on erythrocyte membrane instability under oxidative stress.