Bioinformatic analysis of DOF domains with the aim of selecting a sequence with promising folding behaviour for cloning

Abstract

DOF DNA-binding transcription factor family is a member of zinc finger proteins unique to plants. They are associated with different plant specific phenomena including germination, dormancy, light and defense responses. Until now there is no report of experimentally solved structure for DOF proteins, and obtaining highly purified protein for a member of this family proved to be challenging. This difficulty may in part be related to the strong DNA-binding property of the previously investigated protein. In order to rationally address this problem one may use principles of protein-DNA binding by means of in silico methods to guide the design of experimental approaches. It has been shown that comparative modeling can be used to reliably predict the three-dimensional (3D) model of structurally unknown proteins whenever a suitable template is available. Furthermore, current molecular mechanics force fields allow prediction of interaction energies for macromolecular complexes. Therefore, the approach considered in this work was to model the 3D structures of ZFs from different DOF proteins complexed with DNA molecule, calculate their binding energies, use the energies to select the DOF-ZF with lowest affinity towards DNA, and then clone and express the selected DOF-ZF protein in a suitable host. Materials and Methods: For modeling the ZF domain of DOF3.4, searches at internet resulted in a suitable template with similar amino acid pattern. The alignment of target-template obtained from ClustalW was adjusted manually and used in SWISS- MODEL to generate the model. Then Six DOF members were selected according to their similarity scores and modeled. Binding free energy was calculated for these models using MMPBSA/GBSA methods implemented in amber package. Then the DOF member for cloning was selected according to these calculations, so using the cDNA library prepared from plant Arabidopsis thaliana’ total RNA, the coding sequence for zinc finger domain of selected DOF was cloned into pGEX and transformed into E.coli DH5α. Following the cloning, the DOF-pGEX plasmid was transformed into E.coli BL21 and the expression of the protein was detected by SDS-PAGE gel electrophoresis. Results and Discussion Erythroid transcription factor (GATA1) was selected as the template of modeling. Once the models were built, they were evaluated by Molprobity and Prochek softwares. Among the six members which were modeled and introduced to amber for binding energy calculations DOF5.8 showed the lowest affinity toward its DNA. The corresponding gene was cloned into pGEX vector and transformed into E.coli DH5α. Cloning process was confirmed by sequencing and then the protein was expressed in E.coli BL21 as a GST fusion proteion.

Conclusion: In the current work, a suitable 3D structure was suggested for the zinc finger domain of DOF proteins. A member of this family was cloned and expressed as a fusion protein. This fusion system can be helpful in future purification of this protein with the aim of large scale production of DOF proteins for structural and experimental studies.