Coricello, AdrianaNardone, Alanya J.Lupia, AntonioGratteri, CarmenVos, MatthijnChaptal, VincentAlcaro, StefanoZhu, WenTakagi, YuichiroRichards, Nigel G. J.2025-01-272025-01-272024-12-03Coricello A, Nardone AJ, Lupia A, et al. 3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase. Nat Commun. 2024;15(1):10538. Published 2024 Dec 3. doi:10.1038/s41467-024-54912-9https://hdl.handle.net/1805/45470Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.en-USAttribution 4.0 InternationalCryoelectron microscopyMolecular dynamicsEnzyme mechanismsMolecular conformationProtein structure predictionsArticle