High-resolution structure discloses the potential for allosteric regulation of mitogen-activated protein kinase kinase 7.

Abstract

Mitogen-activated protein kinase kinase 7 (MAP2K7) regulates stress and inflammatory responses, and is an attractive drug discovery target for several diseases including arthritis and cardiac hypertrophy. Intracellular proteins such as MAP2K7 are prone to aggregation due to cysteine-driven oxidation in in vitro experiments. MAP2K7 instability due to the four free cysteine residues on the molecular surface abrogated the crystal growth and led to a low-resolution structure with large residual errors. To acquire a higher resolution structure for promoting rational drug discovery, we explored stable mutants of MAP2K7 by replacing the surface cysteine residues, Cys147, Cys218, Cys276 and Cys296. Single-site mutations, except for Cys147, maintained the specific activity and increased the protein yield, while all the multi-site mutations massively reduced the activity. The C218S mutation drastically augmented the protein production and crystallographic resolution. Furthermore, the C218S crystals grown under microgravity in a space environment yielded a 1.3 Å resolution structure, providing novel insights for drug discovery: the precisely assigned water molecules in the active site, the double conformations in the flexible region and the C-terminal extension bound to the N-terminal region of the adjacent molecules. The latter insight is likely to promote the production of allosteric MAP2K7 inhibitors.

• Authors: Takayoshi Kinoshita, Takuma Hashimoto, Yuri Sogabe, Harumi Fukada, Takashi Matsumoto, Masaaki Sawa
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 493 Issue 1 Pg. 313-317 (11 04 2017) ISSN: 1090-2104 [Electronic] United States
• PMID: 28890347 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2017 Elsevier Inc. All rights reserved.

Chemical References

• MAP Kinase Kinase 7
• MAP2K7 protein, human

Topics

• Allosteric Regulation
• Binding Sites
• Computer Simulation
• Enzyme Activation
• MAP Kinase Kinase 7 (chemistry, ultrastructure)
• Models, Chemical
• Models, Molecular
• Protein Binding
• Protein Conformation
• Structure-Activity Relationship