1 Introduction

Proteins are large molecules formed of building blocks called amino acids. Classical biochemistry commonly says that they are composed of 20 different amino acids, differing in structure from each other; however, currently, this statement is not considered so unequivocally. In addition to the amino acid building blocks, proteins are composed, among others, of the so-called cofactors, i.e., chemical substances other than amino acids, directly bound with proteins that modulate their function. Proteins utilize a large number of different cofactors so that to achieve various structures and properties. One of the most important of these substances are metal ions, which, together with the so-called macromolecules, form metalloproteins, such as metalloenzymes, structural proteins containing metal cores, proteins accumulating metal ions and transporting them. Of all the metal cofactors, transition metal ions play a unique role in the differentiation of function and structure of metalloproteins (Maret & Li, 2009). Among them, the most abundant metal ion in all domains of life is a zinc ion (formally Zn²⁺), which forms with proteins the enormity of structures serving various functions (forming the so-called zinc metalloproteome). Previous studies as well as bioinformatic analysis of the human genome indicate that as many as 10% of the proteins encoded in the genome are zinc proteins (Andreini et al., 2006). This in turn converted into numerical values constituting 3,000 various biomolecules (complexity). Bioinformatic analysis is largely based on sequence homology (signatures), and does not take into account interactions between proteins, which in consequence may mean that there are significantly more zinc proteins than we currently believe (Maret, 2006).

Proteins binding Zn²⁺ ions differ significantly from each other in size, spatial structure, organization into larger complexes or eventually biological function. Despite these differences, the Zn²⁺ ion is bound by proteins using only four amino acid residues, predominantly in the tetrahedral geometry (simplicity). These include cysteine (C), histidine (H), aspartic (D) and glutamic acids (E) residues. The diversity of zinc protein function is not based on a simple combination of these amino acid residues, as this would not lead to the formation of thousands of different molecular forms. This work describes how the zinc enzymes and proteins with zinc ion structural binding sites are modulated by a series of factors leading to the enormous diversity of their function and stability.

2 Modulating Metalloproteins’ Properties via Ligands

Metalloproteins, through specific atom combinations (ligands) involved in the formation of the catalytic or structural site, are able in some way to modulate both their properties and affinity for Zn²⁺. The already classic Pearson’s theory of hard and soft acids says that proteins containing sulfur and nitrogen ligands are substantially more stable than those containing Zn²⁺ ion bound by nitrogen and oxygen ligands (Pearson, 1963). However, a comparison of literature data on the stability of proteins significantly different in terms of binding mode, e.g., alkaline phosphatase (DDHx, DHHx), metallothionein (CCCC) or zinc fingers (CCHH) demonstrates a very similar affinity for Zn²⁺ ions, within the picomolar range (10^–11–10^–12 M). Although the stability of cellular zinc proteins is similar, the thermodynamics of Zn²⁺ binding differs between individual objects (Kochańczyk et al., 2015). Studies conducted on zinc motives showing variable coordination of CCHH, CCHC and CCCC types, with similar affinities, showed different entropic and enthalpic contributions to the free enthalpy of the system (stability). For example, the binding of Zn²⁺ to the CCC motif is driven mainly entropically, which makes this reaction more susceptible to changes in factors such as pH or temperature. With fewer sulfur donors, entropy becomes less favored and the overall stability of the system depends mainly on the enthalpic component (Rich et al., 2012).

On the other hand, proteins containing identical arrangements of ligands associated with Zn²⁺, e.g., HHEx (where x represents a variable external ligand) may exhibit very different affinities for, e.g., prolactin (10⁻⁵ M), angiotensin-converting enzyme inhibitors (10⁻⁸ M) and thermolysin (10⁻¹¹ M). This proves that the so-called first sphere of coordination (Zn²⁺ binding site) is not the main factor affecting the stability of zinc protein. This means that the so-called further protein regions play a key role in the stabilization or destabilization of macromolecules through the interaction of non-binding amino acid residues with each other. Carbonic anhydrase is an example of an extremely thermodynamically and kinetically stable protein, in which the Zn²⁺ ion is bound by three histidine residues (HHHx). Its stability is enhanced by the simultaneous action of ligands binding Zn²⁺ with other amino acid residues in the further regions of the protein through the formation of hydrogen bonds. Mutation of residues involved in the formation of hydrogen bond network, not directly involved in the coordination of Zn²⁺ ions, causes a decrease in the thermodynamic and kinetic stability of the protein (Maret & Li, 2009). Another interesting example of secondary interactions affecting the stability of macromolecules is associated with the simultaneous interaction of ligands binding Zn²⁺ with a peptide bond via hydrogen bonds (-NH⋅⋅⋅S-). One example is the HIV-1 nucleocapsid protein, NCp7 (Namuswe & Berg, 2012).

3 Structural Diversity and Simplicity of Zinc Finger Proteins

The so-called classical zinc fingers show unusual characteristics of structural diversity and at the same time the simplicity of Zn²⁺ binding method. There have been thousands of them identified in different organisms so far, and they are the second class of protein domains in terms of abundance. Despite such a great number, they show high sequence similarity – they contain only approx. 30 amino acids arranged in a highly canonical sequence. The classical zinc finger sequence is as follows: (F/Y)-X-C-X_2-4-C-X₃-(F/Y)-X₅-L-X₂-H-X_3-5-H, where X, M, Y and L represent any amino acid, phenylalanine residue, tyrosine and leucine, respectively (Miłoch & Krężel, 2014). Zn²⁺ ion binds to this motif via two cysteine and histidine residues, forming a characteristic ββα structure responsible in most cases for specific binding to the correct DNA sequence. The metal binding method is identical in thousands of known zinc finger proteins, which has been proven by numerous structural studies. One can safely say that the simplicity of Zn²⁺ ion binding is manifested in the diversity of forms playing a similar function, although specialized towards different molecular targets. Removal of any amino acid responsible for DNA identification in a zinc finger loop results in a very marked decline in the stability of the entire system. Similarly, interference with the so-called hydrophobic cortex of a finger through mutations to less hydrophobic amino acids results in a decrease in stability and a tendency to form other, non-functional structures. Substitution of any donor in a classical zinc finger, e.g., histidine residue to aspartic acid residue, causes dramatic changes in the structure and affinity for Zn²⁺ ion, which is reflected in the functional binding of zinc fingers to DNA (Kochańczyk et al., 2015).

Another relationship between the simplicity and complexity of zinc proteins is exemplified in the presented above stability of zinc domains. The same mode of Zn²⁺ ion binding in the zinc finger domain and a highly conserved sequence would suggest almost identical stability. It turns out that natural zinc fingers are quite diverse in terms of stability, which is perfectly reflected in the so-called consensus zinc finger, being the most representative sequence. The consensus zinc finger contains amino acid residues that occur most frequently at a given position. Its stability is higher than that of natural fingers by more than 1,000 times (Sénèque & Latour, 2010). This is mainly due to a series of interactions occurring in the consensus zinc finger itself, which stabilize the whole structure. Natural zinc fingers do not have that many stabilizing effects within a single structure, which causes a reduction in their stability. Furthermore, the distance between the individual amino acid residues in a sequence of zinc finger also has a significant effect on the modulation of the Zn²⁺ ion affinity. In conclusion, it can be seen that the factually identical metal ion binding mode in a very similar spatial structure (sequence and structure simplicity), as a consequence, manifests itself in different stability and function (stability and function diversity) (Kochańczyk et al., 2015).

4 Complexities of Interprotein Zinc Binding Sites

Although many key zinc proteins have not yet been characterized in terms of affinity for Zn²⁺, definitely the interprotein binding sites of this metal remain the most enigmatic and the least understood. These sites are formed by two or more protein molecules, which bind Zn²⁺ ions together. They both form homo- and heteromer-type structures. Isolation of this type of complexes as well as the characteristics of interprotein binding sites is extremely difficult and available bioinformatic tools are still insufficient to predict them. Isolation of complexes from natural sources or organisms for their overproduction usually results in proteins lacking Zn²⁺ or individual protein units not assembled in higher structures by the metal ion. An example of a protein, in which this type of interaction occurs is RAD50, a part of a highly conserved DNA repair complex – MRE11. This protein forms a homodimer, in which the Zn²⁺ ion is bound by four cysteine residues, each two derived from two identical RAD50 units, to form the so-called hook zinc domain (Stracker & Petrini, 2011). Although the binding of Zn²⁺ by cysteine residues is favored entropically, the formation of a complex of Zn-(protein)₂ stoichiometry becomes unfavorable. There must be other mechanisms present, determining the sufficiently high stability of this site, in order to form this type of complex under physiological conditions. Studies by Kochańczyk et al., 2013 have shown that the formation of an intermolecular hydrophobic network and the formation of hydrogen bonds within peptide chains of the Zn²⁺ binding motif are both responsible for this stabilization (Kochańczyk et al., 2013). Interesting, in terms of complexity and simplicity, is the fact that the zinc hook motif is present in all domains of the living world. It occurs also in the viral proteins. It is not the simplicity of binding of two protein molecules through the coordination of zinc ions that makes it so extremely important, but the manner of maintaining complex stability in organisms, in which Zn²⁺ ion metabolism is highly diversified. As it appears, these are the sequence and structure subtleties (e.g., variations in one or more amino acid residues) that decide whether the protein is stable enough to function, e.g., in thermophilic archeon, or moderately stable as in eukaryotes, so that the kinetics of dissociation and association of Zn²⁺ ions could occur at high speed necessary for the proper functioning of the MRE11 complex.

5 Conclusions

In summary, metalloproteins, and zinc proteins in this particular case, acquire their numerous structures and functions through the binding of small Zn²⁺ ions. The metal-binding method is simple and common to many proteins differing in structure and biological function. Diversification of properties, particularly the thermodynamic stability discussed here, is influenced not only by the bound ligands but also by their additional interactions occurring in other protein regions.