Metal coordination governs the antimicrobial efficacy of calcitermin derivatives.

Antimicrobial peptides are promising alternatives to classical antibiotics. Their microbicidal activity can arise from different mechanisms, one of which is known as nutritional immunity and has metal micronutrients and metal-binding biomolecules as its main players. Calcitermin is an antimicrobial peptide and an effective metal chelator. Its properties as an antibacterial and anti-Candida agent have been recently studied both as a free peptide and in the presence of zinc and copper ions, with which it forms stable complexes. Calcitermin derivatives have also gained attention thanks to the possibility of improving their properties, like metal-binding affinity and/or stability in biological fluids, through ad hoc modifications of the native peptide sequence. In this work, the Ala-to-Ser substitutions close to the coordination site of calcitermin have been introduced to study the impact on the biological activity and metal-binding properties. Our results show that metal coordination has a clear impact on the bioactivity of the studied compounds, to the point that the truncated fragment of calcitermin, solely containing the main metal-binding residues, also shows antimicrobial activity.

Impact of C- and N-terminal protection on the stability, metal chelation and antimicrobial properties of calcitermin.

The main limitation to the use of antimicrobial peptides (AMPs) as regular drugs, against antibiotic and antifungal resistance, mainly relates to their rapid degradation by proteolytic enzymes. The introduction of suitable structural changes in the peptide chain can make the peptide less susceptible to the action of proteases, thus overcoming this problem. To improve the plasma stability of calcitermin, a metal-chelating AMP present in the human respiratory tract and investigated in the present study, C- and/or N- terminal modifications have been introduced in the native sequence. Evaluation of peptide stability has been performed to determine the half-life times in human plasma of both native calcitermin and its derivatives. However, the protection of the peptide termini can also affect its metal coordination behaviour. Thus, the characterization of Zn2+ and Cu2+ complexes has been performed by means of several techniques, including potentiometry, high-resolution mass spectrometry, UV-Vis, circular dichroism and EPR. On the basis of the obtained results, it was possible to compare the biological activity of the studied systems, taking into account both the metal-binding ability and the peptide stability to search for a link among them. A significant result of this study is that the N-terminal protection increases the calcitermin half-life over seven times and the formation of metal complexes confers resistance towards degradation almost doubling its half-life.

Metal-protein solution interactions investigated using model systems: Thermodynamic and spectroscopic methods.

The first-row D-block metal ions are essential for the physiology of living organisms, functioning as cofactors in metalloproteins or structural components for enzymes: almost half of all proteins require metals to perform the biological function. Understanding metal-protein interactions is crucial to unravel the mysteries behind molecular biology, understanding the effects of metal imbalance and toxicity or the diseases due to disorders in metal homeostasis. Metal-protein interactions are dynamic: they are noncovalent and affected by the environment to which the system is exposed. To reach a complete comprehension of the system, different conditions must be considered for the experimental investigation, in order to get information on the species distribution, the ligand coordination modes, complex stoichiometry and geometry. Thinking about the whole environment where a protein acts, investigations are often challenging, and simplifications are required to study in detail the mechanisms of metal interaction. This chapter is intended to help researchers addressing the problem of the complexity of metal-protein interactions, with particular emphasis on the use of peptides as model systems for the metal coordination site. The thermodynamic and spectroscopic methods most widely employed to investigate the interaction between metal ions and peptides in solution are here covered. These include solid-phase peptide synthesis, potentiometric titrations, calorimetry, electrospray ionization mass spectrometry, UV-Vis spectrophotometry, circular dichroism (CD), nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Additional experimental methods, which can be employed to study metal complexes with peptides, are also briefly mentioned. A case-study is finally reported providing a practical example of the investigation of metal-protein interaction by means of thermodynamic and spectroscopic methods applied to peptide model systems.

Investigation of metal interactions with YrpE protein of Bacillus subtilis by a polyhistidine peptide model.

The use of model peptides that can simulate the behaviour of a protein domain is a very successful analytical method to study the metal coordination sites in biological systems. Here we study zinc and copper binding ability of the sequence HTHEHSHDHSHAH, which serves as model for the metal interactions with YrpE, a putative metal-binding protein of the ZinT family identified in Bacillus subtilis. Compared to other ZinT proteins secreted by Gram-negative bacteria, the metal-coordination properties of YrpE N-terminal histidine-rich domain have not been yet characterized. Different independent analytical methods, aimed at providing information on the stability and structure of the formed species, have been employed, including potentiometric titrations, electrospray ionization mass spectrometry, UV-Vis spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopy. The obtained speciation models and equilibrium constants allowed to compare the metal-binding ability of the investigated polyhistidine sequence with that of other well-known histidine-rich peptides. Our thermodynamic results revealed that the YrpE domain HTHEHSHDHSHAH forms more stable metal complexes than other His-rich domains of similar ZinT proteins. Moreover, the studied peptide, containing the alternated (-XH-)n motif, proved to be even more effective than the His6-tag (widely used in immobilized metal ion affinity chromatography) in binding zinc ions.