Towards predicting Ca2+-binding sites with different coordination numbers in proteins with atomic resolution

Abstract

Ca(2+)-binding sites in proteins exhibit a wide range of polygonal geometries that directly relate to an equally-diverse set of biological functions. Although the highly-conserved EF-Hand motif has been studied extensively, non-EF-Hand sites exhibit much more structural diversity which has inhibited efforts to determine the precise location of Ca(2+)-binding sites, especially for sites with few coordinating ligands. Previously, we established an algorithm capable of predicting Ca(2+)-binding sites using graph theory to identify oxygen clusters comprised of four atoms lying on a sphere of specified radius, the center of which was the predicted calcium position. Here we describe a new algorithm, MUG (MUltiple Geometries), which predicts Ca(2+)-binding sites in proteins with atomic resolution. After first identifying all the possible oxygen clusters by finding maximal cliques, a calcium center (CC) for each cluster, corresponding to the potential Ca(2+) position, is located to maximally regularize the structure of the (cluster, CC) pair. The structure is then inspected by geometric filters. An unqualified (cluster, CC) pair is further handled by recursively removing oxygen atoms and relocating the CC until its structure is either qualified or contains fewer than four ligand atoms. Ligand coordination is then determined for qualified structures. This algorithm, which predicts both Ca(2+) positions and ligand groups, has been shown to successfully predict over 90% of the documented Ca(2+)-binding sites in three datasets of highly-diversified protein structures with 0.22 to 0.49 A accuracy. All multiple-binding sites (i.e. sites with a single ligand atom associated with multiple calcium ions) were predicted, as were half of the low-coordination sites (i.e. sites with less than four protein ligand atoms) and 14/16 cofactor-coordinating sites. Additionally, this algorithm has the flexibility to incorporate surface water molecules and protein cofactors to further improve the prediction for low-coordination and cofactor-coordinating Ca(2+)-binding sites.

Figure 1

(A) Calcium ion (sequence ID 2463, green ball) of thermolysin (1FJ3.pdb) from Protein Data Bank (PDB). Binding residues and water oxygen atoms are labeled in the figure. (B) D is a spatial point; b is D-oxygen (red spheres) distance; l is D-carbon (dark grey spheres) distance; θ is D-carbon-oxygen angle; φ is the dihedral angle between the plane formed by the side chain carboxyl group (−COO) and the plane formed by the two carboxyl oxygen atoms (bitentate pair) and D. (C) Calcium ions (sequence ID 8251 and 8252) of serum amyloid P component (1SAC.pdb), its binding residues and cofactor acetic acid.

Figure 2

Schematic diagram of MUG. (A) The extracted oxygen atoms (red spheres) and the constructed graph G. (B) A maximal clique of size five in graph G. (C) The calcium center (blue sphere) of the cluster. The dark gray spheres represent carbon atoms covalently connected to a ligand oxygen atom. (D) Predicted ligand group and Ca²⁺ position.

Figure 3

Flow chart of MUG. Restriction: a cluster must have at least 4 oxygen atoms of specified types where at least n (n= 2, 3, 4) oxygen atoms are from amino acids. Both the type and n are the input parameters.

Figure 4

(A) Distribution of the number of Ca²⁺-binding sites with respect to coordination number of the combined dataset. (B) Population distribution of multiple-binding sites, cofactor-coordinating sites and low-coordination sites in the combined dataset.

Figure 5

Calcium ions and their surrounding atoms for (A) calmodulin (sequence ID 1128, 3CLN.pdb), (B) hcv helicase (sequence ID 6434, 1HEI.pdb), (C) phospholipase A2 (sequence ID 2789, 1PSH.pdb), (D) subtilisin (sequence ID 1943, 1SBH.pdb), and (E) themolysin (sequence ID 2461 and 2462, 1FJ3.pdb).

Figure 6

(A) Distribution of the number of documented sites (black bar) and true predicted sites (gray bar) with respect to coordination number i (1≤ i ≤ 8) (B) Mean deviations (in A) between the documented and predicted sites with respect to coordination number i (1 ≤ i ≤ 8 ).

Figure 7

(A) E-selectin (1ESL.pdb) and calcium ion (sequence ID 1269). (B) Distribution of deviations (in A) (black bars for inclusion of water, red bars for exclusion of water) between the documented and predicted sites with respect to coordination number i (1 ≤ i ≤ 3) for sites that are predicted.

Figure 8

(A) Staphylococcus nuclease (1SNC.pdb), its cofactor tetrahydropyranyl and calcium ion (sequence ID 1084). (B) Distribution of deviations (in A) (black bars for inclusion of cofactor atoms, red bars for exclusion of cofactor atoms) between the documented and predicted sites for sites that are predicted.