title: Seven cysteine-deficient mutants depict the interplay between thermal and chemical stabilities of individual cysteine residues in mitogen-activated protein kinase c-Jun N-terminal kinase 1.
abstract: Intracellular proteins can have free cysteines that may contribute to their structure, function, and stability; however, free cysteines can lead to chemical instabilities in solution because of oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase 1 (JNK1), possesses seven free cysteines and is an important drug target for autoimmune diseases, cancers, and apoptosis-related diseases. To characterize the role of cysteine residues in the structure, function, and stability of JNK1, we prepared and evaluated wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments showed that the chemical stability of JNK1 increased as the number of cysteines decreased. The contribution of each cysteine residue to biological function and thermal stability was highly susceptible to the environment surrounding the particular cysteine mutation. The mutations of solvent-exposed cysteine to serine did not influence biological function and increased the thermal stability. The mutation of the accessible cysteine involved in the hydrophobic pocket did not affect biological function, although a moderate thermal destabilization was observed. Cysteines in the loosely assembled hydrophobic environment moderately contributed to thermal stability, and the mutations of these cysteines had a negligible effect on enzyme activity. The other cysteines are involved in the tightly filled hydrophobic core, and mutation of these residues was found to correlate with thermal stability and enzyme activity. These findings about the role of cysteine residues should allow us to obtain a stable JNK1 and thus promote the discovery of potent JNK1 inhibitors.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Japan.
authors.forename: Tetsuko
authors.initials: T
authors.lastname: Nakaniwa
authors.forename: Harumi
authors.initials: H
authors.lastname: Fukada
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Inoue
authors.forename: Masaki
authors.initials: M
authors.lastname: Gouda
authors.forename: Ryoko
authors.initials: R
authors.lastname: Nakai
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kirii
authors.forename: Motoyasu
authors.initials: M
authors.lastname: Adachi
authors.forename: Taro
authors.initials: T
authors.lastname: Tamada
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Segawa
authors.forename: Ryota
authors.initials: R
authors.lastname: Kuroki
authors.forename: Toshiji
authors.initials: T
authors.lastname: Tada
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Kinoshita
mesh_terms: D000595:Amino Acid Sequence; D003460:Crystallization; D003545:Cysteine / Q000172:deficiency*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects*; D006358:Hot Temperature; D006801:Humans; D048055:Mitogen-Activated Protein Kinase 8 / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D048055:Mitogen-Activated Protein Kinase 8; D003545:Cysteine