The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).

Pb2+ is known to displace physiologically-relevant metal ions in proteins. To investigate potential relationships between Pb2+/protein complexes and toxicity, data from the Protein Data Bank were analyzed to compare structural properties of Pb2+- and Ca2+-binding sites. Results of this analysis reveal that the majority of Pb2+ sites (77.1%) involve 2 to 5 binding ligands, compared with 6 ± 2 for non-EF-Hand and 7 ± 1 for EF-Hand Ca2+-binding sites. The mean net negative charge by site (1.7) fell between values noted for non-EF-Hand (1 ± 1) and EF-Hand (3 ± 1). Oxygen is the dominant ligand for both Pb2+ and Ca2+, but Pb2+ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) Ca2+-binding sites. A comparison of binding geometries where Pb2+ has replaced Ca2+ in calmodulin (CaM) and Zn2+ in 5-aminolaevulinic acid dehydratase (ALAD) revealed protein structural changes that appear to be unrelated to ionic displacement. Structural changes observed with CaM may be related to opportunistic binding of Pb2+ in regions of high electrostatic charge, whereas ALAD may bind multiple Pb2+ ions in the active site. These results suggest that Pb2+ adapts to structurally-diverse binding geometries and that opportunistic binding may play an active role in molecular metal toxicity.

To better understand the biological significance of Ca(2+), we report a comprehensive statistical analysis of calcium-binding proteins from the Protein Data Bank to identify structural parameters associated with EF-hand and non-EF-hand Ca(2+)-binding sites. Comparatively, non-EF-hand sites utilize lower coordination numbers (6 +/- 2 vs. 7 +/- 1), fewer protein ligands (4 +/- 2 vs. 6 +/- 1), and more water ligands (2 +/- 2 vs. 1 +/- 0) than EF-hand sites. The orders of ligand preference for non-EF-hand and EF-hand sites, respectively, were H(2)O (33.1%) > side-chain Asp (24.5%) > main-chain carbonyl (23.9%) > side-chain Glu (10.4%), and side-chain Asp (29.7%) > side-chain Glu (26.6%) > main-chain carbonyl (21.4%) > H(2)O (13.3%). Less formal negative charge was observed in the non-EF-hand than in the EF-hand binding sites (1 +/- 1 vs. 3 +/- 1). Additionally, over 20% of non-EF-hand sites had formal charge values of zero due to increased utilization of water and carbonyl oxygen ligands. Moreover, the EF-hand sites presented a narrower range of ligand distances and bond angles than non-EF-hand sites, possibly owing to the highly conserved helix-loop-helix motif. Significant differences between ligand types (carbonyl, side chain, bidentate) demonstrated that angles associated with each type must be classified separately, and the EF-hand side-chain Ca-O-C angles exhibited an unusual bimodal quality consistent with an Asp distribution that differed from the Gaussian model observed for non-EF-hand proteins. The results of this survey more accurately describe differences between EF-hand and non-EF-hand proteins and provide new parameters for the prediction and design of different classes of Ca(2+)-binding proteins.

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), that predicts Ca 2+ -binding sites in proteins with atomic resolution. After first identifying all 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 highlydiversified protein structures with 0.22 to 0.49 Å 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 cofactorcoordinating 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.

Lead toxicity is associated with various human diseases. While Ca2+ binding proteins such as calmodulin (CaM) are often reported to be molecular targets for Pb2+-binding and lead toxicity, the effect of Pb2+ on the Ca2+/CaM regulated biological activities cannot be described by the primary mechanism of ionic displacement (e.g., ionic mimicry). The focus of this study was to investigate the mechanism of lead toxicity through binding differences between Ca2+ and Pb2+ for CaM, an essential intracellular trigger protein with two EF-Hand Ca2+-binding sites in each of its two domains that regulates many molecular targets via Ca2+-induced conformational change. Fluorescence changes in phenylalanine indicated that Pb2+ binds with 8-fold higher affinity than Ca2+ in the N-terminal domain. Additionally, NMR chemical shift changes and an unusual biphasic response observed in tyrosine fluorescence associated with C-terminal domain sites EF-III and EF-IV suggest a single higher affinity Pb2+-binding site with a 3-fold higher affinity than Ca2+, coupled with a second site exhibiting affinity nearly equivalent to that of the N-terminal domain sites. Our results further indicate that Pb2+ displaces Ca2+ only in the N-terminal domain, with minimal perturbation of the C-terminal domain, however significant structural/dynamic changes are observed in the trans-domain linker region which appear to be due to Pb2+-binding outside of the known calcium-binding sites. These data suggest that opportunistic Pb2+-binding in Ca2+/CaM has a profound impact on the conformation and dynamics of the essential molecular recognition sites of the central helix, and provides insight into the molecular toxicity of non-essential metal ions.