Redox Sensitive Cysteine Residues in Calbindin D28k Are Structurally and Functionally Important†
Abstract
Human calbindin D28k is a Ca2+ binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cysteine-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D28k appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D28k binds Ca2+ with lower affinity than does the reduced protein.
†
This work was supported by NSF Career Grant #9996074, the Lise Meitner Foundation, Lund University and the Camille and Henry Dreyfus Foundation (KSÅ), the Swedish Research Foundation (SL), and the Wallenberg Foundation (TB).
*
To whom correspondence should be addressed. Karin Åkerfeldt: Tel: +1-610-896-1213. Fax: +1-610-896-4963. E-mail: kakerfel@ haverford.edu. Tommy Cedervall: Tel: +46-46-2224470. Fax: +46-46-222 45 43. E-mail: [email protected].
§
Haverford College.
#
Lund University.
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