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 Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans 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.

Physiological and transcriptional profiling of surfactin exerted antifungal effect against Candida albicans

“…The decreased iron content may be part of the general defense against surfactin to minimize the damage caused by ferrous ions (50). In previous studies, the elevated level of free intracellular iron facilitated the production of reactive oxygen species and triggered iron-dependent cell death in Saccharomyces cerevisiae (51). The surfactin-induced reduction in intracellular zinc content was associated with the down-regulation of the ZRT2 gene, which encodes the major zinc importer in C. albicans .…”

Zn2+ and Cu2+ Interaction with the Recognition Interface of ACE2 for SARS-CoV-2 Spike Protein

“…K d does not depend on the ligand concentration, although it depends on the pH. Since K d refers to the general equilibrium ML = M+ L, the lower the value of the constant, the greater the stability of the complex [42,43]. The values of dissociation constants are widely used to compare the stability of the complexes of endogenous ligands.…”

Metal coordination governs the antimicrobial efficacy of calcitermin derivatives

“…Previous studies carried out on metal-chelating peptides have highlighted that the presence of serine residues in the proximity of coordinated histidines favours the binding of the backbone N-amides by copper ions, affecting the complex formation ability of the investigated systems and generally increasing the Cu 2+ binding affinity. [16][17][18] We therefore introduced Ala-to-Ser substitutions close to the coordination site of calcitermin, -HYHTH-, obtaining the following derivatives: VAIALKSAHYHTHKE (A7S), VAIALKASHYHTHKE (A8S) and VAIALKSSHYHTHKE (A7S/A8S), where the alanine residues at positions 7 and/or 8 are replaced by serines. Ala-to-Ser substitutions are introduced with the aim of verifying their impact on metal coordination, and whether this can affect the antimicrobial activity of calcitermin.…”