Structural and functional characterization of Myocyte enhancer factor-2 through evaluation of its DNA-binding ability and virtual screening

Abstract

MEF2 is a family of transcription factors, with four family members; MEF2A, MEF2B, MEF2C and MEF2D, they regulate genetic expression in numerous developmental processes. The highly conserved N-terminal, which contains the MADS and MEF2 domains, is responsible for binding to AT-rich DNA motifs. Despite having similarity in their N-terminal and sharing a similar consensus binding DNA area among all the family members, their functions vary significantly. This study demarcates conformational exploration and comparative binding of DNA specific to their consensus motif YTA(A/ T)4TAR at MADS box/MEF2 domains. α1-helix plays an essential role in MEF2A and MEF2B as it acquire flexibility by attaining the conformation of loop. By comparing it to apo-MEF2, there is an outward positioning of α1-helix distal portion. The formation of hydrophobic groove for the accommodation of DNA by α1-helix to proximal part of β1 allows synergistic repositioning of the α1−α2 linker, C-terminal region, and MEF2 domain. The main contributing factor in both of the cases is the conformational switching of helical content meanwhile conserving the β topology. It remained conserved to maintain the conformation of α1-helix flip. The results of various analyses, such as PCA and DSSP, indicate that MEF2A occupies a greater conformational space during DNA binding compared to MEF2B. Conserved residues, including Arg10, Phe21, and Arg24, are indicative of MEF2-specific residues that play a crucial role in DNA binding. This information led us to structure-based virtual screening to explore novel inhibitors that can inhibit binding of MEF2 with DNA. Acetamide, benzamide and carboxamide top hits that were scrutinized by virtual screening having the ability to inhibit the binding. They were further analyzed by Lipinski’s rule of five, toxicity, absorption, distribution and energy values assessments. On the basis of these findings, active drug like molecules have been proposed against the transcriptional activities of MEF2A and MEF2B. The findings of this study could be instrumental in uncovering the DNA binding mechanisms of MEF2 and may hold significant value in developing therapeutic approaches for MEF2- related disorders. Mutations in transcriptional coactivator myocyte enhancer factor 2B (MEF2B) are known to be most promising causative agent for B-cell non-Hodgkin lymphoma. Molecular pathology behind these causative mutations still needs investigation. In this study we have used molecular DRSML QAU Abstract xii dynamics simulation assays for studying effect of two recurrent mutations (Y69H and K4E) in DNA binding of this important transcription factor. MEF2B has specific N-terminal loop residues (Arg3, Asn113, Gly2, Ile6, Lys4, Lys5) and MADs box domain (Arg24, Lys23 and Lys31). In MEF2BY69H , Ser78, Lys5, Arg3, Arg79 and Asn81 are known to have role in DNA binding, whereas, in MEF2BK4E the residues playing role in DNA binding are Arg3, Arg91 and Gly2. Noticeable conformational changes in α1-N-terminal loop region of MEF2BWT occurs upon DNA binding whereas in MEF2BY69H and MEF2BK4E fluctuations were observed in both α1 and α3. Further analysis includes Hydrogen (H)-bond occupancy that exposes same DNA binding pattern for MEF2WT and MEF2BY69H whereas in case of MEF2BK4E a different pattern has been witnessed. In MEF2BK4E , α1 and α3 has been depicted as the fluctuant regions via Anisotropic Network Model. Residue Tyr69 is involved in p300 binding in all the three complexes. The effect of Y69H variant is also seen in the binding of co-activator p300. This explains reduction in transcriptional activation in case of MEF2BY69H . The current study reveals the structural basis for the recognition of DNA binding by highlighting the essential conformational changes in MEF2BWT, MEF2BK4E and MEF2BY69H dynamics. It may also contribute to identify novel therapeutic plans for lymphomagenesis. MEF2s are transcription factors supervising several cellular activities. MEF2C and MEF2D interact with the E3 ligase F-box protein SKP2, which mediates their subsequent degradation through the ubiquitin proteasome system. The phosphorylation of residues (98 and 110) in MEF2C and -D is an important determinant for SKP2 binding. In this study, through in-silico approaches we have phosphorylated the putative phosphorylation sites (78+98+106+110) in both MEF2C and –D. This study elucidates the conformational patterns, clustered recognition, and differential binding preferences of MEF2C and MEF2D to SKP2 at various phosphorylated sites. In case of MEF2C and –D Sep98+110, N terminal and α1 plays a crucial role whereas in MEF2C and –D Sep98+110 α2 and L4 plays a vital role by acquiring more flexibility and by attaining strong conformation towards α1. By utilizing methods such as docking, molecular dynamics simulations, PCA, and MM-PBSA, this study analyzed the binding patterns and structural characteristics of MEF2C and MEF2D with SKP2. The results showed that all four phosphorylation sites are necessary for the binding of these proteins with SKP2. . Following their involvement in the G0/G1 transition, MEF2C and MEF2D must undergo polyubiquitination and degradation during G1 progression to reduce gene transcription and DRSML QAU Abstract xiii facilitate entry into the S phase. Our results revealed that the processing of MEF2C and MEF2D are depending upon four phosphorylation sites (Ser78, 98, 106, 110). The sequential phosphorylation in MEF2C and –D with SKP2 may be a symbol for authenticating its processing in cell cycle. The influence of individual residue via computational aided methodologies has been speculated in this study. This may be proven vital for the formation of effective and potent inhibitors against MEF2C and MEF2D with the emphasis on the anticancer activities.