Solution Structure Ensembles of the Open and Closed Forms of the ∼130 kDa Enzyme I via AlphaFold Modeling, Coarse Grained Simulations, and NMR

Large-scale interdomain rearrangements are essentialto proteinfunction, governing the activity of large enzymes and molecular machineries.Yet, obtaining an atomic-resolution understanding of how the relativedomain positioning is affected by external stimuli is a hard taskin modern structural biology. Here, we show that combining structuralmodeling by AlphaFold2 with coarse-grained molecular dynamics simulationsand NMR residual dipolar coupling data is sufficient to characterizethe spatial domain organization of bacterial enzyme I (EI), a similar to 130kDa multidomain oligomeric protein that undergoes large-scale conformationalchanges during its catalytic cycle. In particular, we solve conformationalensembles for EI at two different experimental temperatures and demonstratethat a lower temperature favors sampling of the catalytically competentclosed state of the enzyme. These results suggest a role for conformationalentropy in the activation of EI and demonstrate the ability of ourprotocol to detect and characterize the effect of external stimuli(such as mutations, ligand binding, and post-translational modifications)on the interdomain organization of multidomain proteins. We expectthe ensemble refinement protocol described here to be easily transferrableto the investigation of the structure and dynamics of other unchartedmultidomain systems and have assembled a Google Colab page (https://potoyangroup.github.io/Seq2Ensemble/) to facilitate implementation of the presented methodology elsewhere.

Automated summary

Combining structural modeling, molecular dynamics simulations, and NMR data, the spatial domain organization of a bacterial enzyme was characterized, revealing the effect of temperature on its conformational changes. This methodology can be applied to study the structure and dynamics of other multidomain proteins.