4.8 Article

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

Journal

ACS CATALYSIS
Volume 13, Issue 17, Pages 11610-11624

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acscatal.3c02214

Keywords

enzyme; structure of enzyme intermediate; unstableprotein complex; paramagnetic NMR; protein labeling

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We report the solution structure of an authentic sortaseA thioester intermediate with the bound substrate. Through advanced techniques, we determined the 3D structure of this short-lived enzymatic reaction intermediate at atomic resolution. The formation and stability of the intermediate are influenced by calcium binding and its interaction with the substrate, highlighting the importance of calcium in the function of sortase A.
We report the solution structure of an authentic sortaseA thioesterintermediate, including the bound substrate. The structure was determinedby a combination of site-specific tagging, selective isotope labeling,and paramagnetic nuclear magnetic resonance spectroscopy. Pseudocontactshifts generated with different lanthanide tags delivered the conformationof the isotope-labeled bound substrate at atomic resolution, enablingthe determination of the complete 3D structure of the short-livedand lowly populated enzymatic reaction intermediate in solution. Theformation of the unstable thioester complex is accompanied by an increasein calcium binding affinity. Furthermore, the intermediate is significantlystabilized by calcium and decays quickly upon removal of calcium.Cooperativity between calcium binding and substrate recognition thusplays an important role in the function of sortase A.

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