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

• Published in: Biochemical and biophysical research communications Vol. 493; no. 1; pp. 313 - 317
• Main Authors: Kinoshita, Takayoshi, Hashimoto, Takuma, Sogabe, Yuri, Fukada, Harumi, Matsumoto, Takashi, Sawa, Masaaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.11.2017
• Subjects: Allosteric inhibition; Allosteric Regulation; Binding Sites; Computer Simulation; Crystal structure; Cysteine replacement; Enzyme Activation; MAP Kinase Kinase 7 - chemistry; MAP Kinase Kinase 7 - ultrastructure; MAP2K7; Microgravity; Models, Chemical; Models, Molecular; Protein Binding; Protein Conformation; Stable mutant; Structure-Activity Relationship; Allosteric inhibition; Stable mutant; DVD; Cysteine replacement; JNK; MAP2K7; Microgravity; MAP2K; Crystal structure
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2017.09.025

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. •A cysteine mutation stabilized mitogen-activated protein kinase kinase 7 (MAP2K7).•Crystallization under microgravity augmented the crystallographic resolution.•The structure implied the C-terminal region negatively regulated the enzyme activity.•The synthesized C-terminal peptide inhibited the MAP2K7 activity.•The results indicated the potential to produce allosteric inhibitors of MAP2K7.