Calciomics: integrative studies of Ca2+-binding proteins and their interactomes in biological systems

Abstract

Calcium ion (Ca(2+)), the fifth most common chemical element in the earth's crust, represents the most abundant mineral in the human body. By binding to a myriad of proteins distributed in different cellular organelles, Ca(2+) impacts nearly every aspect of cellular life. In prokaryotes, Ca(2+) plays an important role in bacterial movement, chemotaxis, survival reactions and sporulation. In eukaryotes, Ca(2+) has been chosen through evolution to function as a universal and versatile intracellular signal. Viruses, as obligate intracellular parasites, also develop smart strategies to manipulate the host Ca(2+) signaling machinery to benefit their own life cycles. This review focuses on recent advances in applying both bioinformatic and experimental approaches to predict and validate Ca(2+)-binding proteins and their interactomes in biological systems on a genome-wide scale (termed "calciomics"). Calmodulin is used as an example of Ca(2+)-binding protein (CaBP) to demonstrate the role of CaBPs on the regulation of biological functions. This review is anticipated to rekindle interest in investigating Ca(2+)-binding proteins and Ca(2+)-modulated functions at the systems level in the post-genomic era.

Figure 1. Schematics of the Ca²⁺ signaling machinery, the range of Ca²⁺-binding affinities and the timescale of Ca²⁺ modulated activities

The extracellular Ca²⁺ homeostasis is maintained by the coordinated actions of hormones, bone cells and balanced uptake and excretion of Ca²⁺ in intestine and kidney. The internal Ca²⁺ homeostasis is achieved through the exquisite choreography of the Ca²⁺ signaling toolkits. Under resting conditions, cytosolic Ca²⁺ is maintained at submicromolar range by extruding Ca²⁺ outside of the plasma membrane via plasma membrane Ca²⁺-ATPase (PMCA) and Na⁺/Ca²⁺ exchanger (NCX), or by pumping Ca²⁺ back into internal stores through sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) or secretory pathway Ca²⁺-ATPase (SPCA). Upon extracellular stimulation, the free cytosolic Ca²⁺ rapidly increases by the entry of extracellular Ca²⁺ across the plasma membrane via Ca²⁺ channels, including voltage-operated channels (VOC), receptor-operated channels (ROC), transient receptor potential ion-channel (TRP) and store-operated channels (SOC), or by the release of Ca²⁺ from internal stores (e.g., endoplasmic reticulum (ER) and Golgi complex) through inositol-1,4,5-triphosphate receptors (IP3R) and ryanodine receptors (RyR) due to activation of membrane receptors (G protein coupled receptors (GPCRs) and receptor tyrosine kinase [RTK]) and the subsequent synthesis of IP₃. In mitochondria, Ca²⁺ can easily pass through outer mitochondrial membrane pores and cross the inner mitochondrial membrane through the membrane-embedded Ca²⁺ uniporter (MCU). Ca²⁺ exits mitochondria through the opening of a nonselective high-conductance channel permeability transition pore (PTP) in the inner mitochondrial membrane and the Na⁺/Ca²⁺ exchanger (NCX). The Ca²⁺ signals are delivered by affecting the activity of Ca²⁺ buffers, Ca²⁺ effectors and Ca²⁺-regulated enzymes. Ca²⁺-binding proteins have Ca²⁺ affinities that vary by 10⁶-fold or more depending upon their locations and functions (left panel). Ca²⁺ can exert short-term effects by triggering neurotransmitter release within microseconds. The signals can also elicit “long-term” effects by modulating gene expression (right panel).

Figure 2. Profile HMMs for three representative continuous Ca²⁺-binding motifs

The numbers on top of the amino acids indicate the positions of ligands in the Ca²⁺-binding loop. The Pfam entries for EF-hand like (Excalibur), canonical EF-hand and pseudo EF-hand motifs are PF05901, PF00036 and PF01023, respectively. The letter size is positively correlated to the distribution probability of amino acids at each position. See text for detailed discussions (“Section 2.1 Prediction from primary sequences”).

Figure 3. Schematic diagram of approaches used to study calciomics

Starting from protein amino acid sequences, continuous Ca²⁺-binding sites can be simply detected by pattern search, whereas some non-continuous Ca²⁺-binding domains (CaBDs) can be predicted by domain assignment using Pfam. Complementary to these methods, prediction can be made from 3D structural information using several online webservers (e.g., MUG, as listed in Table 1). The proteome for Ca²⁺-binding proteins (CaBPs) can be obtained by standard proteomic approaches coupled with the use of radioactive metals (⁴⁵Ca or ¹⁰³Ru). Quantitative mass spectrometry techniques (e.g., ESI-MS and ICP-MS) can be further applied to characterize the metal/protein stoichiometry of CaBPs. To determine the Ca²⁺-binding affinities, one can use equilibrium dialysis coupled with ICP-MS (or ⁴⁵Ca) under circumstances that the predicted CaBP can be readily produced to high purity while retaining its function. For continuous Ca²⁺-binding sites, a grafting approach can be used to probe metal binding properties without the need to purify the predicted protein. An important aspect of calciomics is to define the CaBP-target interactome by combining both computational and high-throughput experimental approaches. Next, the cellular localization of putative CaBPs can be traced by tagging the CaBP of interest with fluorescent proteins. Lastly, to establish possible functional correlations of Ca²⁺-modulated activities, loss-of-function experiments should be carried out by removing the predicted Ca²⁺-binding ligands.

Figure 4. Prediction of Ca²⁺-binding sites by the geometry and graph (GG) algorithm program

To predict Ca²⁺-binding sites in proteins, oxygen ions (black) from protein 3D-structure are extracted, while other types of ions are excluded. Next, the distance between any of two oxygen ions is calculated, and an edge will be assigned if the calculated oxygen distance is below a cutoff value. A potential Ca²⁺-binding site contains oxygen clusters in which every oxygen ion is linked to each other with an O-O distance less than the cutoff and every oxygen ions is linked to all other oxygen atoms by the assigned edges in this oxygen cluster. The Ca²⁺ center is determined at an equidistant center within each oxygen cluster if the distance ranges from 1.8 to 3.0 Å. Adapted from Deng et al., with permission from John Wiley and Sons.

Figure 5. The interaction of Ca²⁺-CaM with target proteins

Temporal and spatial changes of the Ca²⁺ concentration in different compartments of cells affect the regulation of cellular signaling, by modulating the activity of a large body of CaBPs. Upon binding to Ca²⁺, CaBP such as calmodulin (CaM) undergoes substantial conformational changes and subsequently activate or inactivate over 100 functional enzymes, cellular receptors and ion channels through direct protein-protein interactions.