A distal regulatory strategy of enzymes: from local to global conformational dynamics

Modulating the distribution of various states in protein ensembles through distal sites may be promising in the evolution of enzymes in desired directions. However, the prediction of distal mutation hotspots that stabilize the favoured states from a computational perspective remains challenging. Here, we presented a strategy based on molecular dynamics (MD) and Markov state models (MSM) to predict distal mutation sites. Extensive MD combined with MSM was applied to determine the principally distributed metastable states interconverting at a slow timescale. Then, molecular docking was used to classify these states into active states and inactive ones. Distal mutation hotspots were targeted based on comparing the conformational features between active and inactive states, where mutations destabilize the inactive states and show little influence on the active state. The proposed strategy was used to explore the highly dynamic MHETase, which shows a potential application in the biodegradation of poly(ethylene terephthalate) (PET). Seven principally populated interrelated metastable states were identified, and the atomistic picture of their conformational changes was unveiled. Several residues at distal positions were found to adopt more H-bond occupancies in inactive states than active states, making them potential mutation hotspots for stabilizing the favoured conformations. In addition, the detailed mechanism revealed the significance of calcium ions at a distance from the catalytic centre in reshaping the free energy landscape. This study deepens the understanding of the conformational dynamics of α/β hydrolases containing a lid domain and advances the study of enzymatic plastic degradation.