Quaternary Structure and Lipid Affinity of PLC 85% similarity in sequences are highlighted in black with grey background. Residues present in the X–Y domain and catalytic domain are indicated by black and grey solid bars respectively. Catalytically important and conserved amino acid residues for substrate binding are marked by solid black circles. doi:10.1371/journal.pone.0039258.g001 Catalytic domain of the bacterial (Bacillus thuringiensis) PLC is responsible for dimerization, which possesses only the catalytic XY domain of PLC and also forms a dimer [19,20]. The role of PLC in the unicellular, biflagellate, fresh water alga, Chlamydomonas reinhardtii has been investigated during stress responses [21]. In 1992, Quarmby et. al. also demonstrated the role of PLC in biochemical pathways that couples deflagellation process, induced by the pH shock [22]. However, there is no molecular characterization of the phospholipase from C. reinhardtii. In this study, for the first time we report quaternary structure characterizations, and identify an important disulphide bridge that modulates dimer stability and alters lipid interaction affinity of the novel phospholipase C of Chlamydomonas reinhardtii (CrPLC). CrPLC possesses only catalytic (XY) and C2 domains. The quaternary structure characterization of CrPLC suggests that it exists as dimer in both in vitro and in vivo conditions. Immunolo- calization studies with CrPLC antibody indicate its association with plasma membrane in the Chlamydomonas cells. Results and Discussion Bioinformatics Analysis of the Novel Phospholi Pase from C. reinhardtii Conserved domain analysis of the CrPLC protein sequence (501a.a) suggested the presence of catalytic domain and the C2 domain. Catalytic domain was found to have a significant similarity with that of the eukaryotic phosphoinositide-specific phospholipase C family, which are characterized by the presence of two well conserved histidine residues (H47 and H88) in the catalytic domain. Cluster of hydrophobic amino acid residues, which are responsible in substrate recognition, calcium binding and catalysis of PI-specific PLC [23] were also found to be conserved in CrPLC. These residues includes H47, N48, L69, E77, D79, H88, F110, E122, K164, K166, R286, Y288 and W292 in CrPLC. Domain architecture analysis of CrPLC revealed the presence of catalytic domain that constitutes conserved X and Y regions separated by a linker region and calcium binding C2 domain. Comparison of the deduced amino acid sequence of CrPLC with the other known PLC sequences showed that the conserved residues are mainly present in the catalytic domain and C2 domain (Figure 1). Phylogenetic analysis of CrPLC with the other known phospholipase suggested its close relatedness to phospholipases present in higher plants. Protein sequence align- ment showed overall 45% homology with Arabidopsis thaliana phospholipase (Figures 1 and S1). Predicted tertiary structure of CrPLC resembled an incomplete triose phosphate isomerase (TIM) a/b-barrel structural fold. Catalytic domain in 3-D structure model exhibited alternating a-helices and b-strands whereas calcium binding C2 domain had five antiparallel b- strands arranged as a sandwich. Most of the aromatic amino acid residues including Tyr and Trp present in the tertiary structure of CrPLC was present on the surface of molecule (Figure S2). Thus, overall architecture of the tertiary structure of CrPLC would set an acceptable confirmation of the conserved aromatic rings for further molecular interactions. It could also establish strong hydrophobic interaction with residues from the other CrPLC molecule. CYSPRED server that utilizes the evolutionary infor- mation for prediction of disulphide bonding state of the cysteine, predicted that only Cys7 of CrPLC was in bonded state i.e., likely to participate in the disulphide bond formation whereas other cysteine residues were in non-bonded state (Free State). According to Plant-mPLoc analysis, CrPLC was predicted to be localized in cell membrane. This prediction suggested a possible role of CrPLC in phosphatidylinositol 4, 5-bisphosphate (PIP2) metabolism and lipid signaling pathways in the cell. Recombinant CrPLC Stays as Monomer and Oligomer in Solution The cDNA of CrPLC (1503 bp) was isolated, sequenced and nucleotide sequence encoding CrPLC deposited in GenBank (Accession number JN052078). Recombinant CrPLC was ex- pressed mainly in soluble form and purification level was monitored by SDS-PAGE (Figure S3). CrPLC purified to homogeneity by size exclusion chromatography (SEC) indicated that it was eluted in two fractions (Figure 2A). As determined by calibration of the column with proteins of known molecular standard, fractions 22–25 (elution volume 67–76 ml) eluted at a molecular mass of 110 kDa that corresponded to the apparent molecular mass of CrPLC dimer while fractions 26–29 (elution volume 79–88 ml) corresponded to the molecular mass of CrPLC monomer (55 kDa) (Figure S4). A smaller shoulder observed towards the left of the dimer peak was attributed to the little amount of higher oligomer of CrPLC. Oligomerization in some proteins is known to be affected by dilution (concentration dependent oligomerization) [24]. However, no effect of dilution was observed on dimerization under varying concentration (5 mM to 50 mM) of CrPLC using gel filtration analytical column (Figure 2B). It has also been shown that enzyme activity of some phospholipases was modulated by reversible dimerization of enzyme [25,26]. To assess the monomer-dimer equilibrium of CrPLC, eluted monomer and dimer fractions were reloaded on the preparatory SEC column and eluents were analyzed separately. Reloading of CrPLC monomer and dimer fractions of CrPLC resulted in slight shifting of the monomer elution peaks towards dimer fraction indicating that the purified monomer fraction of CrPLC could form dimer, however no conversion of dimer fraction into monomer was observed, this suggested the existence of a very high-energy barrier between these states (Figures 2C and D) CrPLC monomer was found to form dimer under oxidative conditions, which was not affected by protein dilution, this implied the involvement of strong covalent forces in stabilization of CrPLC dimer. CrPLC Dimerization is Stabilized by Disulphide Bond in Both in vitro and in vivo Conditions Dimerization of recombinant CrPLC was interesting in the light of the fact that phospholipase dimers are known to form functional complexes. Previous studies suggested the role of dimerization as the molecular mechanism by which functional role of phospho- lipases were regulated inside the cell [25]. Chemical crosslinking of CrPLC using glutaraldehyde crosslinking agent, captured dimer of CrPLC as observed on SDS-PAGE (data not shown). Immuno- blotting of chemically cross-linked monomer and dimer fractions using CrPLC specific antibody showed that the monomer fraction was present in both monomer and dimer forms, whereas the dimer fraction was found to be completely cross-linked (dimer). CrPLC dimer in vivo condition was also determined by similar protein PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39258