Zn2+ and Cu2+ Binding to the Extramembrane Loop of Zrt2, a Zinc Transporter of Candida albicans

Abstract

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for <i>Candida albicans</i> zinc uptake and growth at acidic pH. Zrt2 from <i>C. albicans</i> is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126-215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144-153), HxxHxxEHxD (181-193) and the Glu- and Asp- rich sequence DDEEEDxE (161-168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH₂, Ac-DDEEEDLE-NH₂ and Ac-PSHFAHAQEHQDP-NH₂), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn²⁺ and Cu²⁺ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH₂ and Ac-PAHFAHAQEHQDP-NH₂, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu²⁺. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH₂ region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn²⁺ and Cu²⁺ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH₂). All studied Zrt2 loop fragments have lower affinity towards Zn²⁺ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn²⁺ and Cu²⁺ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.