3D Structure of the Transient Intermediate of the Enzyme–Substrate Complex of Sortase A Reveals How Calcium Binding and Substrate Recognition Cooperate in Substrate Activation

We report the solution structure of an authentic sortase A thioester intermediate, including the bound substrate. The structure was determined by a combination of site-specific tagging, selective isotope labeling, and paramagnetic nuclear magnetic resonance spectroscopy. Pseudocontact shifts generated with different lanthanide tags delivered the conformation of the isotope-labeled bound substrate at atomic resolution, enabling the determination of the complete 3D structure of the short-lived and lowly populated enzymatic reaction intermediate in solution. The formation of the unstable thioester complex is accompanied by an increase in calcium binding affinity. Furthermore, the intermediate is significantly stabilized by calcium and decays quickly upon removal of calcium. Cooperativity between calcium binding and substrate recognition thus plays an important role in the function of sortase A.