Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes

Abstract

Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.

A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions

“…These conformational changes result in the exposure of hydrophobic residues, in many cases increasing the binding affinity of calmodulin for target proteins ( Fig. 1 F) (4,20,26). The flexible central linker of calmodulin allows the protein to wrap around the target segment of a binding partner (27)(28)(29)(30)(31).…”

“…A quantitative understanding of this complex network of noncovalent interactions is key to providing fundamental insights into the molecular processes underpinning biological function. A range of biophysical methods have been developed to probe noncovalent protein interactions and the formation of protein complexes (1)(2)(3)(4)(5)(6). However, studies of these processes may be limited by requirements with respect to the quantity of the sample, molecular size, persistence of the labile complexes during the measurement, and resistance of dynamic species to dissociation during sample preparation.…”

“…Surface-based techniques are widely used to detect and characterize protein-protein interactions. Such methods, including surface plasmon resonance (SPR), are convenient, as they can be automated, but they require the attachment of one of the binding partners to a functionalized surface, a process which can affect the binding equilibrium (4,7). Gas phase methods avoid the use of surfaces, and elegant studies of protein complex formation have been made using this approach (2,6), but it can be complex and costly to implement.…”

Optimization of calmodulin-affinity chromatography for brain and organelles

“…Within the Ca 2+ -dependent/-independent (transitional/dual) CaM-BPs we also identified many ribosomal proteins and other proteins associated with ribosomes (31 proteins) like e.g., elongation factor 2, 40S ribosomal protein S3a, 60S acidic ribosomal protein P1, 60S ribosomal protein L13, L19, L22 and L31, which have previously been reported to interact with CaM [63][64][65]. This was unexpected, as Behnen and coworkers showed that CaM binds to ribosome proteins in the presence of Ca 2+ ions and thus plays an important role only in the Ca 2+ -dependent regulation of protein synthesis [63].…”

“…This was unexpected, as Behnen and coworkers showed that CaM binds to ribosome proteins in the presence of Ca 2+ ions and thus plays an important role only in the Ca 2+ -dependent regulation of protein synthesis [63].…”