Membrane Topology and Predicted RNA-Binding Function of the ‘Early Responsive to Dehydration (ERD4)’ Plant Protein

Functional annotation of uncharacterized genes is the main focus of computational methods in the post genomic era. These tools search for similarity between proteins on the premise that those sharing sequence or structural motifs usually perform related functions, and are thus particularly useful for membrane proteins. Early responsive to dehydration (ERD) genes are rapidly induced in response to dehydration stress in a variety of plant species. In the present work we characterized function of Brassica juncea ERD4 gene using computational approaches. The ERD4 protein of unknown function possesses ubiquitous DUF221 domain (residues 312–634) and is conserved in all plant species. We suggest that the protein is localized in chloroplast membrane with at least nine transmembrane helices. We detected a globular domain of 165 amino acid residues (183–347) in plant ERD4 proteins and expect this to be posited inside the chloroplast. The structural-functional annotation of the globular domain was arrived at using fold recognition methods, which suggested in its sequence presence of two tandem RNA-recognition motif (RRM) domains each folded into βαββαβ topology. The structure based sequence alignment with the known RNA-binding proteins revealed conservation of two non-canonical ribonucleoprotein sub-motifs in both the putative RNA-recognition domains of the ERD4 protein. The function of highly conserved ERD4 protein may thus be associated with its RNA-binding ability during the stress response. This is the first functional annotation of ERD4 family of proteins that can be useful in designing experiments to unravel crucial aspects of stress tolerance mechanism

Ribbon model of the putative RNA-binding globular domain.

<p>The ribbon model was constructed by comparative homology approaches. The fold of the domain was identified by fold-prediction meta-server. Due to low pair-wise sequence identity of nearly 10% between the query and identified template, the derived atomic coordinates for the ERD4 globular domain were expected to be of low-resolution. The two ribonucleoprotein motifs (RNP1 and RNP2) in each of the RNA-recognition domains are shown in red and yellow, respectively. The figure was prepared by PyMol (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p

The topology of the <i>B. juncea</i> ERD4 protein.

<p>The toplogy was drawn using TOPO2 tools. The nine transmembrane helices are shown. Also, shown (filled hexagons) is the globular domain containing RNA-recognition domains. The globular domain is suggested to reside inside the chloroplast.</p

Evolutionary relationship among ERD4 homologs.

<p>Evolutionary relationship was inferred using the Neighbor-Joining method in MEGA4 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances are in the units of the number of amino acid substitutions per site. Also shown in brackets are the pair-wise percentage identity between <i>B. juncea</i> ERD4 and other plant proteins, including green algae.</p

Prediction scores for dual organelle targeting of plant ERD4 proteins assessed by ambiguous targeting predictor (APS).

<p>Prediction scores for dual organelle targeting of plant ERD4 proteins assessed by ambiguous targeting predictor (APS).</p

Multiple sequence alignment of plant ERD4 sequences.

<p>The alignment of all available plant ERD4 sequences was achieved using PROMALS3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032658#pone.0032658-Pei1" target="_blank">[42]</a> and only three diverse sequences are shown here. Also shown is the consensus secondary structure predicted by PsiPred; helices are shown as coils and strands are shown as arrows. The nine transmembrane helices are marked as αT. The strictly conserved residues in all the plant ERD4 sequences are shaded, while similar residues are boxed. The residues numbering is of the full-length <i>B. juncea</i> ERD4 protein. The figure was prepared with EsPript suite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032658#pone.0032658-Gouet1" target="_blank">[64]</a>.</p

The best five structural models predicted for the ERD4 globular domain by the fold-recognition servers and their ranking by 3D-Jury method.

<p>(1) PDB identifier code.</p

Amino acid composition of presequences.

<p>Analysis of the amino acid composition of the N-terminal sixteen residues (%MOL-16), N-terminal sixty residues (%MOL-60) and full-length proteins (%MOL-all) (A) analysis of the 123 chloroplast envelope proteins of <i>A. thaliana</i> (B) analysis of plant ERD4 orthologs.</p

Multiple sequence alignment of the ERD4 globular domain.

<p>The alignment was generated by ClustalW. The two RNA-recognition domains are composed of amino acid residues 183–269 (RRM1) and 273–347 (RRM2), respectively. The two ribonucleoprotein motifs of each RRM domain are marked as RNP1 and RNP2. The suggested RNA-interacting residues are marked with filled triangle (▴). The secondary structure elements of each RRM domain in the theoretical structural model are also shown. The strictly conserved residues in all the plant ERD4 sequences are shaded, while similar residues are boxed. The residues numbering is of the full-length ERD4 proteins.</p

Long-term stability of biological denitrification process for high strength nitrate removal from wastewater of uranium industry

The aim of the present study was to biologically denitrify uranium nitrate raffinate (UNR) from nuclear industry, which is a principle source of high strength nitrate waste. To denitrify the high nitrate waste, a pilot-scale continuous stirred tank reactor was designed with two inbuilt settlers. Acclimatization of mixed culture with synthetic waste was carried out prior to the inoculation of the acclimatized sludge into the reactor. Initial concentration of nitrate in uranium raffinate was 77,000 mg/L NO3. It was diluted and used as a feed to the reactor. Concentration of nitrate in feed was increased gradually from 10,000 mg/L NO3 to 40,000 mg/L NO3 with hydraulic retention time (HRT) maintained at 34.4 h. Complete denitrification of 40,000 mg/L NO3 was achieved in a specified HRT. To facilitate understanding of the treatablity and long-term stability of biological denitrification of UNR, study was carried out for 211 days by periodical perturbation of the system. Furthermore, to find the volume ratio of reactor to settler required for the full-scale design of the denitrification plant, settling of acclimatized sludge was carried out