Cryo-EM in Enzymology I:
Structural basis of pH dependence, ligand specificity, and temperature effect

Ming-Daw Tsai / Academia Sinica, Taipei, Taiwan

April 14, 2021, 8 - 9 am (CEST) & 5 - 6 pm (CEST)

Online live talk

Introduction

Mechanistic analysis of enzymes often requires a broad range of specific conditions, but X-ray crystallography is limited by the conditions of crystallization. Cryo-EM can facilitate structural comparison at multiple solution conditions (e.g., pH, ligands, or temperature), which will be addressed with ketol-acid reductoisomerase (KARI) from archaea Sulfolobus solfataricus, which is a dodecamer in solution, displays optimal activity at pH 7-8, and is bi-specific to coenzymes NADH and NADPH. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for KARI:2Mg2+ to address the pH effect.

In addition, cryo-EM structures of two KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, were solved to address the structural basis of cofactor bi-specificity. Most importantly, structures of both the Mg2+-form (KARI:2Mg2+) and its ternary complex (KARI:2Mg2+:NADH:inhibitor) were solved at six temperatures from 4-70° C, leading to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects, and capturing of temperature-resolved intermediates of the temperature-induced conformational change.

 

References

(1) Use of Cryo-EM to Uncover Structural Bases of pH Effect and Cofactor Bi-specificity of Ketol-acid Reductoisomerase. Chen CY, Chang YC, Lin BL, Lin KF, Huang CH, Hsieh DL, Ko TP, Tsai MD. J. Am. Chem. Soc. 141, 6136-6140 (2019). doi: 10.1021/jacs.9b01354

(2) Temperature-resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions. Chen CY, Chang YC, Lin BL, Huang CH, Tsai MD. J. Am. Chem. Soc. 141, 19983-19987 (2019). doi: 10.1021/jacs.9b10687.

Ming-Daw Tsai

Professor Ming-Daw Tsai received B.S. degree from National Taiwan University (1972) and Ph.D. from Purdue University (1978), and served in the faculty of the Department of Chemistry and Biochemistry, The Ohio State University in 1981-2006. Subsequently he moved to the Institute of Biological Chemistry of Academia Sinica, Taiwan. His research interests include mechanistic enzymology of phosphoryl transfer enzymes including DNA polymerases, kinases and phospholipases, and structure-function relationship of proteins in DNA damage response and cancer signaling, including ankyrin repeat proteins and FHA domain proteins. A recent focus is on the roles of TIFA in cancer and immunity. He probes mechanistic problems by applying emerging methodologies in structural biology, including NMR, X-ray crystallography, MS, and recently cryo-EM, leading to close to 300 publications. He was elected to Fellow, American Association for the Advancement of Science (AAAS, 1992), Academician, Academia Sinica (2012), and Fellow, The World Academy of Science (TWAS, 2014).

Contact

The Beilstein Talks are organized by the Beilstein-Institut.
If you have any questions, please contact:

Dr. Carsten Kettner
Beilstein-Institut
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