Experimental and computational approaches for the study of calmodulin interactions

Ca(2+), a universal messenger in eukaryotes, plays a major role in signaling pathways that control many growth and developmental processes in plants as well as their responses to various biotic and abiotic stresses. Cellular changes in Ca(2+) in response to diverse signals are recognized by protein sensors that either have their activity modulated or that interact with other proteins and modulate their activity. Calmodulins (CaMs) and CaM-like proteins (CMLs) are Ca(2+) sensors that have no enzymatic activity of their own but upon binding Ca(2+) interact and modulate the activity of other proteins involved in a large number of plant processes. Protein-protein interactions play a key role in Ca(2+)/CaM-mediated in signaling pathways. In this review, using CaM as an example, we discuss various experimental approaches and computational tools to identify protein-protein interactions. During the last two decades hundreds of CaM-binding proteins in plants have been identified using a variety of approaches ranging from simple screening of expression libraries with labeled CaM to high-throughput screens using protein chips. However, the high-throughput methods have not been applied to the entire proteome of any plant system. Nevertheless, the data provided by these screens allows the development of computational tools to predict CaM-interacting proteins. Using all known binding sites of CaM, we developed a computational method that predicted over 700 high confidence CaM interactors in the Arabidopsis proteome. Most (>600) of these are not known to bind calmodulin, suggesting that there are likely many more CaM targets than previously known. Functional analyses of some of the experimentally identified Ca(2+) sensor target proteins have uncovered their precise role in Ca(2+)-mediated processes. Further studies on identifying novel targets of CaM and CMLs and generating their interaction network - "calcium sensor interactome" - will help us in understanding how Ca(2+) regulates a myriad of cellular and physiological processes.