Minimal functional sites allow a classification of zinc sites in proteins

Abstract

Zinc is indispensable to all forms of life as it is an essential component of many different proteins involved in a wide range of biological processes. Not differently from other metals, zinc in proteins can play different roles that depend on the features of the metal-binding site. In this work, we describe zinc sites in proteins with known structure by means of three-dimensional templates that can be automatically extracted from PDB files and consist of the protein structure around the metal, including the zinc ligands and the residues in close spatial proximity to the ligands. This definition is devised to intrinsically capture the features of the local protein environment that can affect metal function, and corresponds to what we call a minimal functional site (MFS). We used MFSs to classify all zinc sites whose structures are available in the PDB and combined this classification with functional annotation as available in the literature. We classified 77% of zinc sites into ten clusters, each grouping zinc sites with structures that are highly similar, and an additional 16% into seven pseudo-clusters, each grouping zinc sites with structures that are only broadly similar. Sites where zinc plays a structural role are predominant in eight clusters and in two pseudo-clusters, while sites where zinc plays a catalytic role are predominant in two clusters and in five pseudo-clusters. We also analyzed the amino acid composition of the coordination sphere of zinc as a function of its role in the protein, highlighting trends and exceptions. In a period when the number of known zinc proteins is expected to grow further with the increasing awareness of the cellular mechanisms of zinc homeostasis, this classification represents a valuable basis for structure-function studies of zinc proteins, with broad applications in biochemistry, molecular pharmacology and de novo protein design.

Figure 1. Pie charts showing the functions of zinc sites in (A) Zn-superfamilies, and (B) non-redundant zinc proteins (defined as proteins with sequence identity lower than 50%).

The higher proportion of unknown zinc sites and the lower proportion of catalytic zinc sites in (A) with respect to (B) reflect the fact that Zn-superfamilies with unknown functions are generally small (consisting on average of 1.5 non-redundant proteins), whereas those with catalytic functions are generally larger (consisting on average of 7.5 non-redundant proteins).

Figure 2. Zinc ligands found in the representative Zn-sites with structural (left) and catalytic (right) functions, overall (pie charts, top) and as a function of the coordination number (histograms, bottom).

“Other endo” includes all endogenous (i.e., provided by the protein) ligands different from those explicitly indicated, and “Exo” includes all exogenous (i.e., non-protein) ligands. The histogram for structural sites does not take into account the single case of coordination number seven (PDB code 2faw [44]).

Figure 3. Structure and composition of the Zn-clusters identified.

For each cluster, the superimposition of the structures of the representative Zn-sites included in the cluster, the picture of an example structure (shown as a cartoon representation with zinc atoms as blue spheres and zinc ligands as blue sticks), and the list of the representative Zn-sites included in the cluster are given (with the example structure in bold). Each Zn-site is identified by the PDB code and (in parentheses) the residue number(s) and the chain identifier(s) of the zinc atom(s) in the site.

Figure 4. Schematic picture of the positions occupied by zinc ligands in the three subtypes of zinc necklaces.

The occupancy of each position is given as the ratio between the number of sites in which a zinc ligand occurs at that position and the total number of sites belonging to the subtype, and shown as a circle sized proportionally to this ratio. Details on the specific ligands occurring in individual sites are given in Table S4.

Figure 5. Pseudo-clusters grouping part of the unclustered zinc sites.

For each pseudo-cluster, a short description of the criterion used to group the sites, a picture of an example structure (shown as a cartoon representation with zinc atoms as blue spheres and zinc ligands as blue sticks), and a list of the sites included in the pseudo-cluster are given (with the example structure in bold).

Figure 6. Summary charts showing how zinc sites with specific functions are distributed across clusters and pseudo-clusters.

Histogram (A) shows the occurrence of zinc sites with structural, catalytic, regulatory and substrate functions in clusters (left) and pseudo-clusters (right). Pie charts show the shares of structural (B) and catalytic (C) zinc sites occurring in specific clusters and pseudo-clusters, as well as those that remained unassigned (“orphans”). Sectors in pie charts are coloured according to whether clusters and pseudo-clusters contain exclusively or predominantly structural (yellow for clusters and orange for pseudo-clusters) or catalytic sites (red for clusters and purple for pseudo-clusters).