Redox sensitive cysteine residues in calbindin D28k are structurally and functionally important

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Human calbindin D(28k) is a calcium-binding protein involved in cellular protection against apoptosis.
• The protein contains five cysteine residues, whose roles in structure and function are not fully understood.

Purpose of the Study:

• To investigate the structural and functional significance of the five cysteine residues in human calbindin D(28k).
• To explore the impact of cysteine redox state on protein conformation, stability, and biological activity.

Main Methods:

• Site-directed mutagenesis to create cysteine-to-serine mutants.
• Monitoring conformational changes using ANS binding and intrinsic tryptophan fluorescence.
• Assessing protein stability via urea-induced denaturation.
• Mass spectrometry to identify cysteine modifications (glutathione conjugation, sulfenic acid, disulfide-S-monoxides).

Main Results:

• Two N-terminal cysteines undergo redox-driven structural changes, suggesting disulfide bond formation.
• Other cysteines modulate disulfide-dependent conformational changes in a redox-sensitive manner.
• C-terminal cysteines are susceptible to glutathione modification and oxidation to sulfenic acids/disulfide-S-monoxides.
• Oxidized calbindin D(28k) exhibits lower calcium binding affinity compared to the reduced form.

Conclusions:

• The biological function of calbindin D(28k) is intrinsically linked to the redox state of its cysteine residues.
• The N-terminal cysteines are crucial for activating myo-inositol monophosphatase, with enhanced activation under oxidizing conditions.
• Redox modifications of cysteine residues significantly influence calbindin D(28k)'s structure, calcium binding, and enzymatic activity.