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.

Mutation Choice to Eliminate Buried Free Cysteines in Protein Therapeutics

“…Oxidized derivatives of free‐Cys residues, including formation of thiol adducts or cysteic acid, can effectively block refolding—resulting in an irreversible pathway that, by Le Chatelier's principle, will continuously drive protein unfolding. Consequently, buried free‐Cys residues can play a key role in regulating protein functional half‐life …”

“…Conse-quently, buried free-Cys residues can play a key role in regulating protein functional half-life. [4][5][6][7][8] The presence of buried free-Cys residues in protein biopharmaceuticals, notably monoclonal antibodies, is problematic because they can effectively limit shelf-life as well as induce immunogenic aggregates. [9][10][11][12] A recent study of IgG1 and IgG2 antibodies showed that roughly one third of recombinant antibody molecules from a standardized industrial preparation contain a single open disulfide bond (that is, two free-Cys residues that should form a disulfide bond in the native state structure).…”

Impact of cysteine variants on the structure, activity, and stability of recombinant human α‐galactosidaseA

“…Cysteine has unique chemical properties due to its side chain thiol group and can participate in a variety of functional roles such as disulfide bonding, hydrogen bonding and redox regulation . While disulfide bonds play important roles for protein structure and stability, functional roles of free cysteines often depend on their positions and local environment in the protein, and whether it is interior or solvent exposed . We performed a systematic study in this work by replacing each Cys in the rhαGal protein, including the two free Cys, to examine their impact on protein expression, structure, activity and stability.…”

“…In general, free cysteines have been found to play a variety of roles in protein structure and function, including dimerization, metal coordination, enzyme catalysis, redox regulation and thermal stability . The function of the cysteine residue appears to largely depend on its chemical environment within the folded protein structure and has been studied particularly for enzymes involved in lysosomal storage diseases . In rhαGal, the two free cysteines occupy different structural environments.…”

“…14 Since the five pairs of disulfide bonds are distributed in different secondary structures and tertiary folding of the protein with only one pair (Cys142/Cys172) making direct contribution to the active site, 5 it is not clear whether mutations at different Cys positions In general, free cysteines have been found to play a variety of roles in protein structure and function, including dimerization, metal coordination, enzyme catalysis, redox regulation and thermal stability. 15 The function of the cysteine residue appears to largely depend on its chemical environment within the folded protein structure 16,17 and has been studied particularly for enzymes involved in lysosomal storage diseases. 18,19 In rhaGal, the two free cysteines occupy different structural environments.…”