Computational Analysis of EF Hands of PsCBL Protein for Helix and Coil Analysis

The helix/coil tendency of EF-hand stretches was determined by computational analysis using a Chou and Fasman algorithm of ExPASy server (www.expasy.org). EF1 was analyzed, first for its helix motifs. The graphical analysis of EF1 depicts an upward peak for its first 10 amino acids (Fig. 1A) and 20–30 amino acids with an intervening deep peak for 10–20 amino acids, which strongly indicates the high tendency of amino acids (1–10 and 20–30) to form α-helix and a coiled loop region in between. When the same EF1 was viewed for its coil-forming propensity (Fig. 1B), amino acids 10–20, which showed a deep peak in case of helix analysis, showed a sharp upward peak in case of coil/loop analysis, indicating the high tendency for these amino acids to form a loop structure. Thus EF1 had the necessary helix-loop-helix motifs, which is the characteristic feature of EF hand. The same was also true for EF hand 2, 3 and 4 (Fig. 1A–D). Thus PsCBL had all the functional domains, which is a prerequisite for calcium binding activity for this protein.

Homology Based Modeling of PsCBL and OsCBL Using AtCBL2 Structure as a Template

The crystal structure of AtCBL2 has been determined at 2.1 A resolution (Nagae et al, 2003). PsCBL shares 89% identity at amino acid level with AtCBL2, therefore AtCBL2 was used as a template for modeling PsCBL as well as OsCBL, (92% identical with PsCBL). Structural predictions were made using Swiss-model server and visualized using Rasmol software. is the ribbon diagram of AtCBL2 and Figure 2B and C are the structural predictions of PsCBL and OsCBL, respectively. The polypeptide chain of all these predicted protein structures are folded into two globular domains (N and C terminal) composed of α-helical structure with 9 α-helices (shown in pink) and four short β-strands (in yellow). The loop region between the two α-helices is the calcium binding site and the β-strands stabilize the structure of protein. High degree of conservation is seen between the protein structures of AtCBL, PsCBL and OsCBL. There was no significant difference in the overall topology and protein folding, when these structures were overlapped. Figure 2D and E are the overlapped structures of AtCBL2 with PsCBL, and PsCBL with OsCBL, respectively.

Therefore, the homology based modeling represents high degree of conservation between AtCBL, PsCBL and OsCBL. This also reflects that calcium-binding proteins perform important function and necessitates the conservation of their overall 3 dimensional structures in different species. The number of calcium ions coordinated depends on the sequence of the protein and structural characteristics favor the coordination of calcium ions in particular EF hand motifs. AtCBL2 for example has two-calcium loaded EF hands, EF1 and EF4, whereas the second and third EF hand motifs are maintained in the open form by internal hydrogen bonding without coordination of calcium ions.12 The amino acid sequences of all the EF hands of PsCBL exactly match with AtCBL2. Therefore, it is expected that PsCBL may also coordinate calcium ions in the second and fourth EF hand as seen for AtCBL2. However, this statement still awaits experimental validation.

Homology Based Modeling of PsCIPK and AtCIPK18 Using Chk1 Structure as Template

Check point kinase (Chk1) is a serine-threonine kinase that plays an important role in the DNA damage response, including G₂/M cell cycle control. The crystal structure of the Chk1 kinase domain has been determined.13 Part of CIPKs is similar to Chk1 and this similarity is interesting as sos2 mutants show cell cycle defect at the root meristem in the presence of Na⁺ stress.14 However, there has been no further report unraveling the fundamental behind this observation. Nevertheless, AtCIPKs are characterized by their unique NAF motif, required for CBL interaction. When PsCIPK sequence was submitted to Swiss-model server, predictions were made using Chk1 as a template. Structural prediction for only 275 amino acid residues of PsCIPK from the N terminal could be possible. is the ribbon diagram of Chk1 kinase domain. Chk1 kinase domain is composed of two subdomains, an 88-residue N-terminal domain and a 210-residue C-terminal domain. The N-terminal domain is composed largely of β-structure, the C-terminal domain is largely helical, and are connected by an extended linker region. The catalytic site is at the junction between these two domains.

The structural predictions of PsCIPK and AtCIPK18 were made using Chk1 structure as a template. Figure 3B and C are the predicted 3D structures of PsCIPK and AtCIPK18 respectively. High degree of conservation is seen between Chk1, PsCIPK and AtCIPK18 for the part for which the structure is known (till 275 amino acid residues of PsCIPK). These proteins were almost identical and there was no significant difference in the overall topology and protein conformation, when these structures were overlapped. Figure 3D and E are the overlapped structures of Chk1 with PsCIPK, and PsCIPK with AtCIPK18 respectively. The β-structure N-terminal domain is in yellow, largely helical C-terminal domain in pink and the activation loop is in green. The activation loop in all these proteins was between the conserved amino acids APE and DFG. The activation loop of Chk1 is stable and folded into an open and active-like conformation. On the basis of predicted model the activation loop of PsCIPK and AtCIPK is also folded into an open and active-like conformation in the absence of phosphorylation or ligand.

Direct Interaction of PsCBL with PsCIPK Mutants Via Yeast Two-Hybrid System

Further, T184D and deletion FISL mutants of PsCIPK were constructed. The interaction of PsCBL with PsCIPK mutants, were checked in vivo by yeast 2-hybrid system. is the template for panel B to D showing the clones streaked: Yeast (AH109) cells containing cotransformants of AD-PsCIPK (cloned under EcoR I and Xho I sites) and BD-PsCBL (cloned under Nco I and EcoR I), clone 1 (the positive control for this experiment), 2. cotransformants of AD-PsCIPK T184D plus BD-PsCBL, 3 AD-PsCIPKT184D and BD vector alone, 4. cotransformants of AD-PsCIPKΔFISL and BD-CBL, 5. cotransformants of AD-PsCIPKΔFISL and BD vector alone, 6. cotransformant of AD-PsCIPK T184DΔFISL and BD-CBL, 7. cotransformants of AD-PsCIPK double mutant and BD vector alone, 8. cotransformant of BD-CBL and AD vector alone, 9. cotransformants of AD-CIPK and BD vector. All these transformants grew well on the on SD-Leu^-Trp^- medium (2 drop out medium) Fig. 4B. In a selection medium lacking Leu, Trp and His (SD-Leu^-Trp^-His^-+15 mM 3-AT) only selected clones of cotransformants (BD-PsCBL plus AD-PsCIPK and the AD-PsCIPKT184D plus BD-CBL), in which the HIS3 gene was transactivated, showed growth The results from β-galactosidase filter lift assay for colonies of cotransformants giving blue color further confirmed the interaction between PsCBL and PsCIPK (full-length) and between PsCBL and PsCIPKT184D proteins panel 1 and 2 respectively). This result shows that PsCIPK mutant with the deletion in its auto-inhibitory (FISL/NAF) motif, failed to interact with PsCBL thus confirming the authenticity of these proteins and revealing the significance of FISL domain as an important interaction module required for PsCBL and PsCIPK interaction.