Metallothionein-3 and carbonic anhydrase metalation properties with Zn(II) and Cd(II) change as a result of protein–protein interactions

Abstract

Metallothioneins (MT) are regulators of the metals Zn(II) and Cu(I) and act as antioxidants in many organisms, including humans [1–3]. Isoform 3 (MT3) is expressed constitutively in central nervous tissue and has been shown to have additional biological functions, including the inhibition of neuronal growth, the regulation of apoptosis, and cytoskeleton modulation. To facilitate these functions, protein-protein interactions likely occur. These interactions may then impact the metalation status of the MT and the recipient metalloprotein. Using electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report that the interaction between the zinc metalloenzyme, carbonic anhydrase (CA), and MT3, impacts the metalation profiles of both apo-MT3 and apo-CA with Cd(II) and Zn(II). We observe two phases in the metalation of the apo-CA, the first of which is associated with an increased binding affinity of apo-CA for Cd/Zn(II) and the second pathway is associated with apo-CA metalated without a change in binding affinity. The weak interactions that result in this change of binding affinity are not detectable as a protein complex in the ESI-mass spectral data or in the circular dichroism spectra. These unusual metalation properties of apo-CA in the presence of apo-MT3 are evidence of the effects of protein-protein interactions. With adjustment to take into account the interaction of both proteins, we report the complete Cd(II) and Zn(II) binding constants of MT3 under physiological conditions, as well as the pH dependence of these binding pathways.