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BackgroundG protein-coupled receptors (GPCRs) constitute a large family of integral transmembrane receptor proteins that play a central role in signal transduction in eukaryotes. The genome of the protochordate Ciona intestinalis has a compact size with an ancestral complement of many diversified gene families of vertebrates and is a good model system for studying protochordate to vertebrate diversification. An analysis of the Ciona repertoire of GPCRs from a comparative genomic perspective provides insight into the evolutionary origins of the GPCR signalling system in vertebrates.ResultsWe have identified 169 gene products in the Ciona genome that code for putative GPCRs. Phylogenetic analyses reveal that Ciona GPCRs have homologous representatives from the five major GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin) families concomitant with other vertebrate GPCR repertoires. Nearly 39% of Ciona GPCRs have unambiguous orthologs of vertebrate GPCR families, as defined for the human, mouse, puffer fish and chicken genomes. The Rhodopsin family accounts for ~68% of the Ciona GPCR repertoire wherein the LGR-like subfamily exhibits a lineage specific gene expansion of a group of receptors that possess a novel domain organisation hitherto unobserved in metazoan genomes.ConclusionComparison of GPCRs in Ciona to that in human reveals a high level of orthology of a protochordate repertoire with that of vertebrate GPCRs. Our studies suggest that the ascidians contain the basic ancestral complement of vertebrate GPCR genes. This is evident at the subfamily level comparisons since Ciona GPCR sequences are significantly analogous to vertebrate GPCR subfamilies even while exhibiting Ciona specific genes. Our analysis provides a framework to perform future experimental and comparative studies to understand the roles of the ancestral chordate versions of GPCRs that predated the divergence of the urochordates and the vertebrates.

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Calnuc is a novel, highly modular, EF‐hand containing, Ca2+‐binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G‐proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G‐proteins, specifically with the α‐subunit. Herein, we report the structural implications of Ca2+ and Mg2+ binding, and illustrate that Calnuc functions as a downstream effector for G‐protein α‐subunit. Our results show that Ca2+ binds with an affinity of 7 μm and causes structural changes. Although Mg2+ binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca2+ binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G‐protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca2+ homeostasis in the cell. Using Stains‐all and terbium as Ca2+ mimic probes, we demonstrate that the Ca2+‐binding ability of Calnuc is governed by the activity‐based conformational state of the G‐protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Giα. With the number of organelle‐targeted G‐protein‐coupled receptors increasing, intracellular communication mediated by G‐proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G‐proteins.

Calumenin (Calu) is a well-conserved multi-EF-hand-containing Ca-binding protein. In this work, we focused on the alterations that calumenin has undergone during evolution. We demonstrate that vertebrate calumenin is significantly different from its invertebrate homologues with respect to its response to Ca binding. Human calumenin (HsCalu1) is intrinsically unstructured in the Ca free form and responds to Ca with a dramatic gain in structure. Calumenin from Caenorhabditis elegans (CeCalu) is structured even in the apo form, with no conformational change upon binding of Ca. We decode this structural and functional distinction by identifying a single "Leu" residue-based switch located in the fourth EF-hand of HsCalu1, occupied by "Gly" in the invertebrate homologues. We demonstrate that replacing Leu with Gly (L150G) in HsCalu1 enables the protein to adopt a structural fold even in the Ca free form, similar to CeCalu, leading to ligand compensation (adoption of structure in the absence of Ca). The fourth (of seven) EF-hand of HsCalu1 nucleates the structural fold of the protein depending on the switch residue (Gly or Leu). Our analyses reveal that the Leu that replaced Gly from fishes onward is absolutely conserved in higher vertebrates, while lower organisms have Gly, not only enlarging the scope of Ca-dependent structural transitions but also drawing a boundary between the invertebrate and vertebrate calumenin. The evolutionary selection of the switch residue strongly corroborates the change in the structure of the protein and its pleiotropic functions and seems like it can be extended to the presence or absence of a heart in that organism.

Calcium binding proteins (CBPs) function in response to changes in intracellular calcium (Ca 2+ ) levels by modulating intracellular signaling pathways. Calcium sensors, including Nucleobindins (Nucb1/2) undergo Ca 2+ -binding induced conformational changes and bind to target proteins.Nucleobindins possess additional uncharacterized domains including partly characterized EFhands. We study the molecular evolution of Nucleobindins in eukaryotes emphasizing on the Nterminal DNA binding domain (DBD) that emerged as a result of domain insertion event in Nucb1/2 domain-scaffold in an ancestor to the opisthokonts. Our results from in silico analyses and functional assays revealed that DBD of Nucb1 binds to canonical E-box sequences and triggers cell epithelial-mesenchymal transition (EMT). Thus, post gene duplication, Nucb1 has emerged as unconventional Ca 2+ -binding transcriptional regulators that can induce EMT. Highlights:1. Nucleobindins emerged from prokaryotic EF-hand containing protein.2. The DNA-binding domain was gained in these in opisthokonts.3. Gene duplication in ancestor of euteleostomes, lead to emergence of Nucb1/2. Nucb1 emerged as a canonical E-box binding protein promoting EMT

BackgroundGold nanoparticles (AuNP) are extensively used as biophysical tools in the area of medicine and technology due to their distinct properties. However, vivid understanding of the consequences of biomolecule-nanomaterial interactions is still lacking. In this context, we explore the affect of conjugation of Gαi1 subunit (of heterotrimeric G-proteins) to AuNP and examine its consequences. We consider two bio-conjugation strategies covalent and non-covalent binding.ResultsAffinity of the AuNP to the Gαi1 is 7.58 × 10 12 M-1. AuNP conjugated Gαi1 exhibits altered kinetics of activation, non-covalent bio-conjugates displays retarded kinetics, up to 0.88 fold when GTPγS was used as ligand, of protein activation contrary to covalent conjugates which accelerates it to ~ 5 fold. Conjugation influence intrinsic Gαi1 GTPase function in conflicting modes. Non-covalent conjugation inhibits GTPase function (decrease in activity upto 0.8 fold) whilst covalent conjugation drastically accelerates it (12 fold increase in activity). Altered basal nucleotide uptake in both types of conjugates and GTPase function in non-covalent conjugate are almost comparable except for GTPase property of covalent conjugate. The effect is despite the fact that conjugation does not change global conformation of the protein.ConclusionThese findings provide clear evidence that nanoparticles, in addition to ‘passive interaction’ with protein (biomolecule), can interact “actively” with biomolecule and modify its function. This concept should be considered while engineering nanoparticle based delivery systems in medicine.

Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP-independent class of phospholipid translocators which possess a single EF-hand-like Ca 2+ -binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca 2+ for ΔCTH-hPLSCR1 (K a = 115 lM) compared with hPLSCR1 (K a = 249 lM). Far UV-CD studies revealed that the CTH peptide adopts a-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOX-CAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca 2+ coordination and also plays an important role in the functional conformation of hPLSCR1.