Metallothioneins (MTs)， small cysteine-rich proteins， present in four major isoforms， are key proteins involved in zinc and copper homeostasis in mammals. To date， only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M4S11 (α) and M3S9 (β) clusters. Recent discoveries indicate that Zn(ii) ions are bound with MT2 with the range from nano- to picomolar affinity， which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(ii)-depleted MT2 species. These forms serve as Zn(ii) donors or acceptors and are formed under varying cellular free Zn(ii) concentrations. Due to the lack of appropriate methods， knowledge regarding the structure of partially-depleted metallothionein is lacking. Here， we describe the Zn(ii) binding mechanism in human MT2 with high resolution with respect to particular Zn(ii) binding sites， and provide structural insights into Zn(ii)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis， MALDI MS， bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the α-domain is formed sequentially in the first stages， followed by the formation of the β-domain， although both processes overlap， which is in contrast to the widely investigated cadmium MT. Independent ZnS4 cores are characteristic for early stages of domain formation and are clustered in later stages. However， Zn-S network rearrangement in the β-domain upon applying the seventh Zn(ii) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(ii) ion associates with the β-domain by coordination to Cys21， which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(ii) binding to the isolated β-domain differs significantly from the whole protein， which explains its previously observed different Zn(ii)-binding properties. MD results obtained for Zn(ii) binding to the whole protein and isolated β-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis.