Dynamics of flexible intracellular regions modulate communication and fine-tune pH gating in plant aquaporins

Intracellular pH regulation of PIP2 aquaporins, primarily mediated by a histidine sensor in loop D, is essential for controlling water transport. While the C-terminal domain is involved, the precise contribution of N-terminal and other flexible regions has been debated. Given that PIPs are small proteins that lack large regulatory domains yet achieve finely tuned gating, we sought to investigate how local structural flexibility and inter-region communication contribute to this process. To this end, we introduced alanine substitutions into conserved proline motifs in BvPIP2;2, targeting a contiguous N-terminal PPP motif (BvPIP2;2-AAA) and two isolated prolines in loop D (BvPIP2;2-P194A and BvPIP2;2-P204A). Using heterologous expression in Xenopus laevis oocytes, we evaluated channel function and localization, while the structural impact of the mutations was analyzed with molecular dynamics simulations on full-length structural models. While BvPIP2;2-P204A impaired membrane trafficking, both BvPIP2;2-AAA and BvPIP2;2-P194A mutants localized to plasma membrane and remained functional. Functional assays revealed that the BvPIP2;2-AAA mutant exhibited a marked alkaline shift in pH0.5, greater than that observed for BvPIP2;2-P194A. Molecular dynamics simulations showed that the N-terminal mutation reduced flexibility, reorganized the network of interactions between N- and C-terminal regions, and altered the environment and the pKa of the canonical pH sensor, His202. In contrast, the P194A mutation produced more modest structural and functional changes. These findings support a model in which transitions in structural flexibility within intracellular regions, particularly the N-terminal, govern communication between dynamic segments and modulate the electrostatic environment of the pH sensor, thereby fine-tuning pH gating in plant aquaporins.