Despite the improvement of antifungal therapies over the last two decades and the extensive research devoted to the development of new therapeutic strategies, there is currently only a limited number of effective drugs to fight fungal infections and the phenomenon of drug resistance is still of major concern in clinical practice.[1] One of the biggest obstacles in finding effective, pathogen-specific therapeutics arises from the fact that both fungi and mammals belong to Eukaryota domain and, thus, they share essential metabolic pathways.[2] In order to design highly specific antifungal drugs, it is crucial to understand and aim at the differences in the metabolism of humans and pathogens. Although fungus-selective targets are scarce, one significant difference between fungal and mammalian cells concerns on the transport systems of zinc ions. In fact, several studies have shown that zinc acquisition and regulation greatly contribute to both virulence and physiology of certain pathogenic species, like C. albicans.[3] In the present work, we focus on the zinc uptake and vacuole storage controlled by Cot1, a transmembrane protein composed of 199 amino acid residues and mainly involved in the transport of cobalt and zinc ions.[4] Since Cot1 structure is still unsolved, Phyre2,[5] a web-based service for protein structure prediction, was employed. The most probable Zn(II) binding domain of Cot1 was identified at its C-terminal region, i.e. in the extracellular part of the protein (Fig. 1). The most interesting fragment is an outer-cellular loop containing histidine-rich sequences, which is quite well conserved among zinc-binding proteins from fungal species. The Zn(II) binding behavior towards three peptides Ac-FHEHGHSHSHGSGGGGGG-NH2 (residues 131-148), Ac-SHSHSHSHS-NH2 (residues 157-165), and Ac- FHEHGHSHSHGSGGGGGGSDHSGDSKSHS HSHSHS-NH2 (residues 131-165), corresponding to the putative binding domain of Cot1 at the C-terminal region, was investigated. We also decided to study Cu(II) interactions, since copper is a good reference metal for Zn(II) as well as a necessary nutrient for C. albicans. High-resolution mass spectra were recorded using a time-of-flight mass analyzer (TOF) with an electrospray ionization (ESI) source spectrometer, in order to obtain useful information about stoichiometry of the formed species at a given pH. Stability constants for proton, Zn(II) and Cu(II) complexes were calculated from pH/metric titrations. Finally, spectroscopic techniques, such as UV-Vis, Circular Dichroism and Electron Paramagnetic Resonance, provided information about the complex-structure and coordination modes of the investigated peptides. [1] Pettit, N. N.; Carver, P. L. Ann. Pharmacother. 2015, 49 (7), 825-842. [2] Vandeputte, P.; Ferrari, S.; Coste, A. T. Int. J. Microbiol. 2012, 2012:713687. [3] Morschhäuser, J. Fungal Genet. Biol. 2010, 47 (2), 94-106. [4] Conklin, D. S.; McMaster, J. A.; Culbertson, M. R.; Kung, C. H. I. N. G. Mol. Cell. Bio. 1992, 12 (9), 3678-3688. [5] Kelley, L. A.; Mezulis, S.; Yates, C. M.; Wass, N. M.; Sternberg, M. J. Nat. Protoc., 2015, 10 (6), 845-858.
The zinc binding ability of Cot1, a metal transporter from Candida albicans
Denise Bellotti
;Maurizio Remelli;
2017
Abstract
Despite the improvement of antifungal therapies over the last two decades and the extensive research devoted to the development of new therapeutic strategies, there is currently only a limited number of effective drugs to fight fungal infections and the phenomenon of drug resistance is still of major concern in clinical practice.[1] One of the biggest obstacles in finding effective, pathogen-specific therapeutics arises from the fact that both fungi and mammals belong to Eukaryota domain and, thus, they share essential metabolic pathways.[2] In order to design highly specific antifungal drugs, it is crucial to understand and aim at the differences in the metabolism of humans and pathogens. Although fungus-selective targets are scarce, one significant difference between fungal and mammalian cells concerns on the transport systems of zinc ions. In fact, several studies have shown that zinc acquisition and regulation greatly contribute to both virulence and physiology of certain pathogenic species, like C. albicans.[3] In the present work, we focus on the zinc uptake and vacuole storage controlled by Cot1, a transmembrane protein composed of 199 amino acid residues and mainly involved in the transport of cobalt and zinc ions.[4] Since Cot1 structure is still unsolved, Phyre2,[5] a web-based service for protein structure prediction, was employed. The most probable Zn(II) binding domain of Cot1 was identified at its C-terminal region, i.e. in the extracellular part of the protein (Fig. 1). The most interesting fragment is an outer-cellular loop containing histidine-rich sequences, which is quite well conserved among zinc-binding proteins from fungal species. The Zn(II) binding behavior towards three peptides Ac-FHEHGHSHSHGSGGGGGG-NH2 (residues 131-148), Ac-SHSHSHSHS-NH2 (residues 157-165), and Ac- FHEHGHSHSHGSGGGGGGSDHSGDSKSHS HSHSHS-NH2 (residues 131-165), corresponding to the putative binding domain of Cot1 at the C-terminal region, was investigated. We also decided to study Cu(II) interactions, since copper is a good reference metal for Zn(II) as well as a necessary nutrient for C. albicans. High-resolution mass spectra were recorded using a time-of-flight mass analyzer (TOF) with an electrospray ionization (ESI) source spectrometer, in order to obtain useful information about stoichiometry of the formed species at a given pH. Stability constants for proton, Zn(II) and Cu(II) complexes were calculated from pH/metric titrations. Finally, spectroscopic techniques, such as UV-Vis, Circular Dichroism and Electron Paramagnetic Resonance, provided information about the complex-structure and coordination modes of the investigated peptides. [1] Pettit, N. N.; Carver, P. L. Ann. Pharmacother. 2015, 49 (7), 825-842. [2] Vandeputte, P.; Ferrari, S.; Coste, A. T. Int. J. Microbiol. 2012, 2012:713687. [3] Morschhäuser, J. Fungal Genet. Biol. 2010, 47 (2), 94-106. [4] Conklin, D. S.; McMaster, J. A.; Culbertson, M. R.; Kung, C. H. I. N. G. Mol. Cell. Bio. 1992, 12 (9), 3678-3688. [5] Kelley, L. A.; Mezulis, S.; Yates, C. M.; Wass, N. M.; Sternberg, M. J. Nat. Protoc., 2015, 10 (6), 845-858.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
