Fig 1.
Structural comparison of Gh-CML11 with other CML members in Nicotiana benthamiana and Arabidopsis thaliana.
Calcium dependent and calcium independent structural variations for Gh-CML11 protein with A. NbrsgCaM and B. AtCML38.
Fig 2.
Identification of CaMB motifs in CLCuMB-βC1 sequence and structure.
A. Selected CaM binding motifs are highlighted in CLCuMB-βC1 protein showing potential binding capacity with Gh-CML11. B. Deletion of CaMB motif 1-5-8-14 reduces ΔΔG value suggesting its role in binding at positions 66–79 in CLCuMB-βC1 α-helix (highlighted with purple color). C. Most known canonical CaMB motifs listed here are present in CLCuMB-βC1, indicating its potential interaction with calmodulin like protein in Ca+2-dependent manner.
Table 1.
Possible canonical CaM binding motifs in CLCuMB-βC1.
Fig 3.
Interaction prediction using protein docking methods and machine learning method using 3D structures of CLCuMB-βC1 and Gh-CML11 protein.
A. Top ten models predicted by docking methods ZDOCK and DOCKING2 were aligned within 5 Å, highlighted and most common residues involved in binding site were selected. Red color shows binding residues in Gh-CML11 and blue color indicates interacting residues in CLCuMB-βC1. Black arrow represents a closer view of single model among ten models. B. Machine learning method PRISM itself evaluated interacting residues within 6 Å for interaction, and interacting amino acids from PRISM are highlighted in red and blue color. C. Another machine learning method PAIRPred retrieves data from both sequence and structure. Output result produces graphical illustration of interacting residues between Gh-CML11 and CLCuMB-βC1. Color from orange to red indicates interacting region in both proteins based on B-factor.
Fig 4.
Cumulative score for binding site between CLCuMB-βC1 and Gh-CML11 protein based on sequence and structure prediction methods.
A. Graphical illustration shows binding site results at residue level. CLCuMB-βC1 residues from 60–75 position in α-helix and residues at 103–106 position has high binding affinity for interaction. Consensus of all computational methods shows Gh-CML11 possess higher binding affinity in the central part (residues at position 60–77) and C-lobe (residues at position 144–150) respectively. B. Predicted residues are shown in both virus and host protein structures.
Fig 5.
Interaction between Gh-CML11 and CLCuMB-βC1 using yeast two hybrid system and pull down assay.
A. Positive colonies on yeast agar media [SD-Ura-His-Trp (+L) and SD-Ura-His-Trp-Leu (-L)] indicated interaction between Gh-CML11 and CLCuMB-βC1. Interaction was further enhanced with serial dilution of 3-AT that confirmed the in-silico results for Gh-CML11 and CLCuMB-βC1. Interaction of IYSV-TSWV coded N proteins were assessed as a positive control in y2h system. Gh-CML11 fused with empty prey vector was used as a negative control during autoactivation step. B. Gh-CML11 protein cloned in pMAL-c2X/MBP was purified with CLCuMB-βC1 protein present in pDEST15/GST-tagged. Purification steps includes load samples that represents crude extracts, wash sample shows removal of unbound proteins and elution samples represents band for positive interaction for Gh-CML11 and CLCuMB-βC1. IYSV-TSWV N proteins were used as a positive control and Gh-CML11 transformation with empty prey was used as a negative control.
Fig 6.
Subcellular localization of Calmodulin like protein Gh-CML11 interaction with CLCuMB-βC1 protein.
Gh-CML11 with CLCuMB-βC1 were cloned into GATEWAY compatible BiFC vectors for interaction analysis. For protein localization, Nicotiana benthamiana leaves was used, carrying cyan fluorescent protein fused with nuclear specific histone marker 2B (CFP-H2B). A. Interaction between Gh-CML11 with CLCuMB-βC1 was localized in plasma membrane. B. Guard cells fluorescein with cyan color indicates localization in the cytoplasm. C. Negative control showing successful transformation protocol. Images were obtained 48h post-infiltration at 20x+1.5 zoom option. Scale bar = 50 μm.