Characterization of the Metal-Binding Sites of Metallothioneins and Structural Changes Between Metal-Free and Metal-Bound Forms by Ion Mobility-Mass Spectrometry (IM-MS)

Abstract

Metal ions play important roles in many chemical and biochemical processes, such as oxidation, oxygen transport, and electron transfer. The cellular trafficking of metal ions is controlled by a group of low molecular weight (6-10 kDa), cysteine-rich proteins (∼30%) referred to as metallothioneins (MTs). Despite intensive research of MTs for more than 50 years, the precise physiological function of MTs has not yet been identified. X-ray crystallography and NMR studies of fully cadmium-saturated human MTs have revealed two binding domains in Cd7-MT: the N-terminal β-domain Cd3(Cys)9 and the C-terminal α-domain Cd4(Cys)11. Although the structure of fully-metallated MT was published 30 years ago; the structures of metal-free and partially-metallated MTs are extremely difficult to determine and the metal-binding mechanism remains unclear. To understand the structural change of MT upon metal binding, we performed titration experiments and ion mobility-mass spectrometry (IM-MS). Titration experiments revealed preferential formation of Cd4-MT with addition of <4 eq Cd2+, suggesting the cooperative binding of four Cd2+ to MT. IM-MS ATD for apo-MT are composed of a single, broad peak, consistent with multiple conformers, possibly disordered structure, whereas the ATD for Cd7-MT is composed of 2-3 distinct peaks. Moreover, shifts in the ATD as MT accumulates metals, suggest the formation of more compact structures upon binding of metals. The results from these studies underscore the potential of IM-MS for monitoring structural changes that occur as the number of metals ions bind to MT, both in terms of increasing and decreasing metal content. In addition, proposed conformers of partially and fully metallated species and preferences for metal ion binding can be examined by using combining ion mobility with tandem mass spectrometry and hydrogen/deuterium exchange (HDX) chemistry.