Wheat, a member of the Poaceae family is a cardinal food crop. Wheat conjointly with other staple cereals contributes to high amount of calories and nutrients. Hexaploid wheat (Triticum aestivum L. AABBDD) is the essential food crop for more than 1/3^rd of the global population. It is an important source of major micronutrients. The present day wheat consisting of around 42 chromosomes lacks several important micronutrients like zinc and iron [1].

The NAM-B1 gene, a NAC transcription factor was the first gene to be held accountable for the variation in grain protein content. This was identified using a map-based positional cloning approach in wheat. The allele for high grain protein content was identified in a wild emmer accession [2]. Wheat plays an essential role in the translation machinery. It is also responsible for maintaining the nutritional seed grain quality in wheat [2]. This gene plays an efficient role in various processes such as development, DNA Binding, leaf senescence and protection against stresses [3].

NAM-B1 delayed senescence by three weeks affected the Fe and Zn concentrations in grain [4]. The Sequence comparison between hexaploid and tetra ploid wheat varieties, reflected that most modern wheat varieties have non-functional protein either due to deletion, frame shift mutation within the first intron or a thymine residue insertion at position [5]. Tetraploid wheat consists of NAM-B1 functional orthologous copy on chromosome 6A characterizes as Gpc-A1. One paralogous copy is present on chromosome 2B, represented as Gpc-B2. Hexaploid wheat Gpc-1 has two functional orthologous copies on chromosomes 6A and 6D (Gpc-D1) and two paralogous Gpc-2 copies that are present on chromosomes 2B (Gpc-B2) and 2D (Gpc-D2). The admittance of the wild emmer NAM-B1 gene into modern wheat cultivars result in near-isogenic lines with up to 20% higher nutrition value especially Zn and Fe.

TaNAM-B1 in bread wheat is accountable for their role in seed and grain developmental processes, etc. but little is known about their structure. Development of 3D model of the same would help us in better understanding and enhancing the grain and nutritional quality of crop. In the present investigation, in silico analysis and homology modelling studies TaNAM-B1 from Bread wheat was reported as the three dimensional structures of this gene were not developed. Hence for the better understanding of the structural feature and molecular functions, the model structure for the respective gene was constructed. The model TaNAM-B1 was validated with PROCHECK, PROSAII, Verify3D, RMSD. Homology modeling of TaNAM-B1 could prove beneficial in functional characterization of plant response to improve the nutritional grain quality and leaf senescence.

Screening protein sequence

The sequence of the Triticum aestivum TaNAM-B1 was retrieved from NCBI database URL http://www.ncbi.nlm.nih.gov/ with accession number AIZ97665.1. Template selection was done using BLASTp for the query against PDB [6]. The target subsequently selected was the x ray crystal structure with PDB code 3ULX. Sequence alignments were completed employing ClustalW. ScanProsite was used to identify motif.

Alignment and phylogenetic analysis

All the sequences of APX were aligned using ClustalW http://www.ebi.ac.uk/Tools/msa/ method to find out the similarity present among the sequences of the same family. Phylogenetic analysis of the sequences was done MEGA (version 4.0.02) [7], using UPGMA method. Each node was tested using the bootstrap approach by taking 5,000 replications. The number is indicated in percentages against each node. The branch lengths were drawn to scale indicated.

Motif analysis

Protein motifs analysis was done by online (Multiple Expectation Maximization for Motif Elicitation) tool version 3.5.7 method by using width of motif 25 and 50 residues respectively and number of 7 motifs, remaining rest of default parameters [8].

Secondary structure prediction

Analysis of secondary structure prediction of the TaNAM-B1 protein sequences we employed several programs based on different algorithms such as PSSFinder, prediction using Markov chains, PSIPRED and EMBL (http://www.ebi.ac.uk/services/structures) protein structure tool.

Structure modeling and validation

Structure was modeled by method of homology SWISS-MODEL and by ‘threading’ method (I-TASSER). Energy minimization was done employing the Swiss-PDB-Viewer with potential forces [9]. The 3D models were checked by Verify 3D. Steric and conformational were assessed employing Ramachandran plot server ProCheck and ProSA-Web. In RMSD, corresponds to distance between two objects [7]. This value showed the degree to which two 3D structures are similar. The RMSD value between the template 3ULX and our model structure was calculated employed MOE.

A total of 18 full-length amino acid sequences of NAM-B1 from different plants were considered for multiple sequence alignment. Multiple sequence alignment highlighted in blue colour represents the conserved sequence of amino acid residues among homologues of NAM-B1 families. This conservation is related with dissimilarities which are adequate for carrying out variations at the structural and functional levels (Figure 1). For phylogenetic analysis among NAM-B1 from various plants, a rooted tree was generated using the respective sequences (Figure 2). The phylogenetic analysis of NAM-B1 with different plants indicated two clusters: Cluster A, and cluster B. Cluster A showed two different sub-cluster (a) and (b). Its tree upon the expansion, give NAM-B1 in Aegilops speltoides, Aegilops longissima, Aegilops bicornis, Triticum aestivum, Aegilops tauschii, Triticum timopheevii, Triticum dicoccoides, Hordeum vulgare, Brachypodium distachyon, Oryza sativa, Setaria italic, Zea mays, Sorghum bicolor, Vigna gracilis, Manihot esculenta and Manihot nana Müll. Arg which indicated orthologous and homologous linkage amongst themselves. This signified that NAM-B1 protein family is strictly conserved from ancestral plants. A broad exploration of protein motifs and their function was done using MEME tools, which recognized various conserved motifs in the NAM-B1 (Table 1 and Figure 3). Protein motifs studies also confirm similar essentials for improvement of this NAM-B1 family. Motifs consisting of the signature are well conserved and having substitutions which do not affect their activity. A total of seven protein motifs: 1, 2, 3, 4, 5, 6 and 7 were clearly observed amongst 18 different NAM-B1 sequences present widely amongst plants. In all plant, motif-1 was most commonly observed and was functionally related to Casein kinase II phosphorylation site, N-myristoylation, Protein kinase C, LDL-receptor class B and NAC domain properties. Motif-2 present, was functionally related to N-myristoylation site, NAC and NAM profile. Motif-3 was related to CK2_PHOSPHO_SITE Casein kinase II phosphorylation site, NAC domain profile isoforms. Motif 4 represented Protein kinase C phosphorylation site, NAC. Beside this, Motif-5, 6 and 7 were most commonly related to Casein kinase II, N-glycosylation site, and Histidine-rich region with respect. With a high probability, 7% alpha helices and 15% beta sheets were found in the molecule. The existence of other secondary structure elements and their length could not be determined unambiguously (Figure 4). The length of TaNAM-B1 protein sequences was found to be 404aa residues by sequence search using BLAST identified with PDB ID: 3ULX. The OsNAM-B1 (PDB ID: 3ULX) showed 59.2% sequence identity to the query sequence with an e-value of 4e-69. ScanProsite server predicted that the fragment ranging from 34- 205 residues was NAC domain, ID PS51005. The sequence alignment revealed that the NAM-B1 Transcription DNA binding stress responsive domain was conserved both in TaNAM-B1 as target and OsNAM-B1 as template (Figure 5). These highly conserved transcription factor performs dual function under biotic and abiotic stress responses generated during plant growth and development. The structure was then predicted and taken further for validation. The stereochemical quality and accuracy of the predicted TaNAM-B1 model was evaluated by procheck tool using Ramachandran plot. It revealed 93% residues were found in most favored region, 17% residues in additionally allowed region, 5% residues in generously allowed and 3% residues were found in the disallowed region following the Ramachandran plot. This model was compared with the Swiss Model and 95.0% residues were found in the most favored region, 3% residues in additionally allowed region and 2% residues in generously allowed region. Consequently the model structure was further produced employing Molecular Modeling and Simulations tools (MOE) which was used for the analysis. To further analyze the overall model quality of target (TaNAM-B1) and template (OsNAM-B1), we compared using ProSA web analysis server and this further revealed a Z score value measure of model quality and total energy of the both structures such as template and target as -4.74 of OsNAM-B1 and -4.95 of TaNAM-B, respectively. The Z-score analysis revealed a decent accuracy of our TaNAM-B1. Verify3D was used for determination of the compatibility of an atomic model (3D) with its own residues by assigning a structural class based on its alpha, beta, and loop, polar, nonpolar. Therefore, the comparison result revealed 79.89% of the residues had a score ≥0.2 for a best quality of our TaNAM-B1 model. The degree of structure similarity was measured using root-mean- square distance (RMSD) between corresponding pair of atoms as template (1.483 Å) and target (1.32 Å). The RMSD examination of the wheat TaNAM-B1 model was measured using its template and employing web server tools like SuperPose.

Figure 1: Multiple sequences alignment of deduced amino acid from TaNAMB-1 and other homologues protein sequences.

Figure 2: Phylogenetic tree analysis showing similarity of TaNAMB-1 with other plants using MEGA software version 4.0.

Figure 3: Conserved motif sequences in the deduced amino acid sequences of NAM-B1 from wheat and other homologues.

Figure 4: Secondary structure prediction of the beta strand and alpha helix topology of TaNAMB1 protein sequences.

Figure 5: Schematic cartoon representation: (A) Structural Modeling of TaMAB1 visualized by PyMOL; and (B) Representation of N-C terminal using Discovery Studio V.4.1.

Table 1: Multilevel consensus and their functions for the MEME defined motifs.

NAC transcription factor NAM-B1 of the NAC family is associated with the grain protein content. It is key factor involved in the regulation of gene expression and plays a significant role in the improvement of the nutritional quality of seed. This study focused on the development of the 3D structure of TaNAM-B1 which would facilitate further study in research. This model developed would act as a good source for functional analysis of crystal structure derived experimentally. The motifs analyzed during the study can be further altered using genome editing tools, such as CRISPR-cas9 technology. This would help us in various genomic advancements resulting in better molecular function in seed grain quality, nutritional value and abiotic stress tolerances to crops.