Comparison of the solution conformations of human [zn₇]-metallothionein-2 and [cd₇]-metallothionein-2 using nuclear-magnetic-resonance spectroscopy

The solution structure of native human [Zn₇]-metallothionein-2 has been compared with the previously determined structure of human [Cd₇]-metallothionein-2. The comparison was based on complete sequence-specific ¹H nuclear magnetic resonance assignments for human [Zn⁷]-metallothionein-2 obtained using the sequential assignment method. The secondary structure was found to be very similar in the [Zn₇]- and |Cd₇]- forms of the protein. Only seven amide protons in [Zn₇]- metallothionein-2 were found to have exchange rates lower than ~0.2 min⁻¹ at pH 7.0 and 10 °C, which corresponds closely to the results of amide proton exchange studies with the [Cd₇]- form of the protein. Finally, the ¹H-¹H distance constraints determined from nuclear Overhauser enhancement spectroscopy for human [Zn₇]-metallothionein-2 were checked for compatibility with the [Cd₇]-metallothionein-2 structure. Overall, although no direct method is available for identifying the metal polypeptide co-ordinative bonds in the Zn²⁺-containing protein, these measurements provided several independent lines of evidence showing that the [Zn₇]- and [Cd₇]- forms of human metallothionein-2 have the same molecular architecture.11 page(s

Three-dimensional structure of human [¹¹³Cd₇]metallothionein-2 in solution determined by nuclear-magnetic-resonance spectroscopy

The three-dimensional structure of human [¹¹³Cd₇]metallothionein-2 was determined by nuclear magnetic resonance spectroscopy in solution. Sequence-specific ¹H resonance assignments were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with heteronuclear correlation spectroscopy, ¹H-¹H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling constants ³JHNα and Jαβ. The molecule consists of two domains, the β-domain including amino acid residues 1 to 30 and three metal ions, and the α-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.15 page(s

Amide proton-exchange in human metallothionein-2 measured by nuclear magnetic resonance spectroscopy

In human metallothionein-2, the exchange rate constants of ten amide protons were found to range from 1.7 × 10⁻⁴ to 1 × 10⁻¹ min⁻¹ at pH 6.3 and 8 °C. Most of these slowly exchanging protons could be associated with hydrogen bonds in secondary structure elements of the α-domain. Amide proton exchange rates thus present an additional criterion for the structural characterization of different metallothioneins, which could be particularly valuable for comparisons of different homologous protein preparations containing nuclear magnetic resonance-inactive metal ions, where the metal-polypeptide co-ordinative bonds cannot be identified directly.6 page(s