Systematic transition modeling analysis in the MEF2B-DNA binding interface due to Y69H and K4E variants
Graphical abstract
Introduction
The two most common forms of mature B-cell lymphoid neoplasms are diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) that account for over 50% of all diagnoses [1,2]. Both lymphomas are derived from B-cells at the germinal center (GC) stage of differentiation. The gene encoding myocyte enhancer factor 2B (MEF2B) is somatically mutated in approximately 15% of DLBCL and FL cases [[3], [4], [5], [6], [7], [8], [9]], and a small fraction (~3%) of mantle cell lymphomas [9]. In human, 4 MEF2 paralogs (MEF2A, MEF2B, MEF2C and MEF2D) exist that are involved in the endothelial cell regulation of muscle, neural crest, lymphocyte development, chondrocyte and neuron [10]. MEF2B is the most distant family member of MEF2 family that consists of an N-terminal DNA-binding MADS domain, a central MEF2 domain, and a C-terminal transcriptional activation domain. So far, two isoforms of MEF2B have been described, designated as isoform-A and B that differ at their transcriptional activation domains [11] and neither isoforms share 31% identity of amino acids with any other MEF2 proteins [12]. An additional uniqueness of MEF2B is a change in the MADS domain, whereby a glutamic acid residue, fundamental for DNA binding activity is substituted by a glutamine (E14Q). MEF2B is well known for its binding to DNA in dimeric form illustrated in nervous system, heart problems, muscle disorders and cancerous diseases explained in Giorgio et al. [13]. Subsequent analysis by Lei et al. [14] described similar physiochemical properties and DNA binding residues for both dimeric and monomeric forms of MEF2B.
MEF2B dysfunction has been associated in a variety of human diseases including neurodegeneration, heart failure and cancer [15]. The distinctive features of MEF2B compared to the other family members hold true for cancer [13]. More recently, MEF2B and MEF2C have been reported to be highly mutated in the major forms of non-Hodgkin lymphomas (NHLs). NHLs include 8–18% diffuse large B-cell lymphoma (DLBCL) [3,5,16,17], 13% Follicular lymphoma (FL) [16] and 3–7% mantle cell lymphoma (MCL) [9,18]. Morin et al. analyzed MEF2B mutations present in NHLs in the context of MEF2BWT crystal structures [16,19,20]. NHLs are highly heterogeneous as various MEF2B mutations contribute to lymphoma development via unknown mechanisms.
In MEF2B, K4, Y69 and D83 residues located in MADS and MEF2 domains are important for DNA binding and dimerization. Majority of the mutations, including K4E and D83V generally reduce DNA binding and transcriptional activity of MEF2B [12]. In DLBCL, MEF2B hot spot mutations (K4 and Y69) occur in the regions solely required for dimerization and DNA binding. Using ectopically expressed MEF2B, Pon and coauthors identified MEF2B regulated genes in kidney 293 cells and demonstrated that K4E and Y69H mutations reduce MEF2B transcriptional activity [12].
The peripheral region of MEF2B contains more β-branched residues that favor β-strand conformation [21]. This region together with the C-terminal of the MEF2 domain (82–91 residues) forms an α-helix (H3). Structural analyses reveal that by facilitating extensive hydrogen bonding and electrostatic interactions between N-terminus of H3 and β3-strand, D83 plays a central role in stabilizing the helical conformation of H3. MEF2BD83V uniquely contributes in the induction of substantial and specific structural changes in the MEF2 domain. MEF2BD83V structural analysis revealed that the conformation switch of helix H3 leads to the loss of interactions between DNA and a cluster of positively charged residues on H3 [19]. This structural change is consistent with electrophoretic mobility shift assay results by Pon et al., demonstrating that MEF2BD83V binds less efficiently to DNA than MEF2BWT [12]. The binding of CABIN1 and possibly other co-factors such as class IIa HDACs is controlled by the allosterically structural stability of β3-strand [19]. In MEF2BWT, β3 structure is stabilized by D83-mediated interactions with helix H3. The β3/H3 region has also been implicated in the interaction with other transcription factors that synergistically act together with MEF2, such as MyoD (myoblast determination protein 1) [22]. D83V mutation may affect the binding of these MEF2 co-factors. Finally, D83V-induced structural changes may create new sites for protein-protein interactions that may promote the assembly of diverse set of MEF2 transcriptional complexes and help in the recruitment of new transcription co-factors.
MEF2 is a major partner of p300 in muscle, neurons and T-cells [[22], [23], [24], [25], [26]]. It turns on and off gene expression in a calcium-dependent manner [27]. In the resting state, MEF2 inhibits the expression of target genes by recruiting the co-repressors to specific loci of the genome [28,29]. When activated, the co-repressors dissociate from MEF2 through calcium-dependent mechanisms [30]; the DNA-bound MEF2 further interacts with calcium-activated transcription factors (e.g. NFAT and CREB) and recruits other co-activators such as p300 and myocardin to activate transcription [31,32]. p300 also directly regulates the transcriptional activity of MEF2 by controlling the acetylation of MEF2 [33,34]. The overall transcription state of MEF2-bound promoters is tightly controlled by signal-dependent protein-protein interactions among MEF2 and co-regulators. The interaction between MEF2 and its co-activator p300 reveals a sequence-specific recruitment of transcription co-activator p300 by the DNA-bound transcription factor [35]. Here, we investigate the comparative DNA binding preferences of MEF2BWT, MEF2BK4E and MEF2BY69H and underlying conformational changes through in silico approaches. Our current study may assist in uncovering the impact of most frequent MEF2B mutations leading to lymphomagenesis and data may be helpful in devising effective strategies for NHL therapeutics.
Section snippets
Data collection
The primary phase included the collection of point mutations involved in non-Hodgkin's Lymphoma. To classify the nature (pathological or neutral) of mutations, PROVEAN (Protein Variation Effect Analyzer) [36], IMutant 2.0 [37] and MutPred [38] servers were employed to predict the impact of mutations on the protein stability. PROVEAN predicts the functional effect of single or multiple amino acid substitutions, insertions and deletions using the prediction tool. The server provides rapid
Sequence and structural analysis
Based on the findings of PROVEAN, IMutant 2.0 and MutPred servers (Table S1), 2 MEF2B point mutations (K4E and Y69H) having role in lymphomas [12] were selected for detailed structural analysis. The three dimensional structures of MEF2BK4E and MEF2BY69H were modeled through homology modeling using MEF2B crystal structure (PDB ID: 1N6J) as a template. The structures were minimized by employing conjugate gradient algorithm and Amber force field in UCSF Chimera 1.11.2 [18]. Ramachandran scores for
Discussion
MEF2 TFs play diverse roles in growth and adaptive reactions. Despite their well-characterized roles, mechanism by which MEF2 TFs regulate transcriptional activity remains impenetrable. MADs-box domain has been selected by numerous proteins that bind equally well to their target DNA in a highly bent form as for SRF (Serum Response Factor) and MCM1 (pheromone receptor transcription factor) [66] or to relatively unbent DNA as for MEF2A [67]. MADS-box domain exhibits certain variable residues on
Accession codes
1N6J MEF2B.
3P57 p300 TAZ2 domain bound to MEF2.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We acknowledge members of Functional Informatics Lab, National Center for Bioinformatics for their indispensable support and encouragement.
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