Crystal Structure of Apo MEF2B Reveals New Insights in DNA

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Crystal structure of Apo MEF2B reveals new insights in DNA binding and cofactor interaction Xiao Lei, Haoran Shi, Yi Kou, Niroop Rajashekar, Fang Wu, Chandani Sen, Jiang Xu, and Lin Chen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00439 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 28, 2018

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Biochemistry

Crystal structure of Apo MEF2B reveals new insights in DNA binding and cofactor interaction Xiao Lei1, Haoran Shi1, Yi Kou1-3, Niroop Rajashekar1, Fang Wu4, Chandani Sen1, Jiang Xu1-3, and Lin Chen1-3* 1

Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA 2

Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA

3

USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA

4

Department of Statistics and Applied Probability, University of California, Santa Barbara, CA 93106, USA

KEYWORDS MEF2; MADS box superfamily; Self-regulatory; Protein-DNA recognition; Transcription factor; Cancer Mutation Supporting Information Placeholder ABSTRACT: The myocyte enhancer factor 2 (MEF2)

family of transcription factors play important roles in developmental processes and adaptive responses. Although MEF2 proteins are known to bind DNA in the nucleus to regulate specific gene expression, there are reports that MEF2 also functions in the cytoplasm. Previous structural studies of MEF2 focused exclusively on DNA-bound MEF2 with and without various corepressors or co-activators. While these studies have established a comprehensive structural model of DNA recognition and co-factor recruitment by MEF2, the structure of MEF2 not bound to DNA, which include cytoplasmic MEF2 and free MEF2 in the nucleus, is unknown. Here we determined the structure of the MADS-box/MEF2 domain of MEF2B without DNA nor cofactor. The Apo structure of MEF2B reveals a largely preformed DNA binding interface that may be important for recognizing the shape of DNA from the minor groove side. In addition, our structure also reveals that the C-terminal helix of the MEF2-specific domain could flip up to bind to the hydrophobic groove that serves as the binding sites of MEF2 transcription cofactors. These observations shed new insights into DNA binding and cofactor interaction by MEF2 proteins.

Introduction Myocyte enhancer factor 2 (MEF2) is a family of transcription factors with important roles in cardiac and skeletal development, T cell activation, memory formation and hematopoietic cell differentiation 1,2. There are four members of MEF2 in human, MEF2A, MEF2B, MEF2C and MEF2D. The full length MEF2 proteins consist of 360 to 520 amino acids residues that can be divided into the MADS box domain, the MEF2 specific domain, and the transcriptional activation domain. The highly conserved N-terminal MADS box and MEF2-specific domains (amino acids residues 1 to 90) are responsible for dimerization, DNA binding and co-regulator interaction (Figure S1). MEF2B was originally found to be highly expressed in B cells and recently also found to be frequently mutated in lymphoma 3–6. MEF2 is predominantly located in the cell nucleus; at least a fraction of nuclear MEF2 was constitutively bound to DNA to control gene repression or activation depending on the presence of co-activator (e.g. p300, FAK) or co-repressor (e.g. Cabin1, class II HDACs) in a given cell state 1. The MEF2-specific domain is required for interaction with cofactors. Deletion of and mutations in this domain lead to loss of or decreased cofactor binding, including MyoD, Cabin1, HDAC4, and FAK 7–11. The MEF2-specific domain was

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reported to be unstructured in earlier crystallography and NMR studies 12,13, but later was found to adopt a betastrand and an alpha helix on the side of the MADS-box core in a number of binary MEF2/DNA or ternary MEF2/DNA/cofactor complexes 8,14,15. In addition to extensive studies of MEF2’s function in the nucleus, there is evidence that cytoplasmic MEF2 also has physiological importance, especially in brain function and memory formation 16–19. Previous structural studies of MEF2/DNA/cofactor complexes have shed lights on how MEF2 recognize DNA and cofactors. In this study, we determined the structure of MEF2B in the absence of DNA or any cofactor (designated Apo MEF2B). Our studies reveal two important insights into the structure and function of MEF2. First, the apo structure of MEF2 is similar to its DNA-bound state except for the Nterminal tails. The major DNA binding surface, which consists of helix H1 and its exposed residues, are preformed and may impose the binding specificity at the level of DNA shape recognition and base-specific interactions. Another unexpected finding is the ability for the alpha helix of the MEF2B specific domain (helix H3) to fold up and bind the hydrophobic groove of the cofactor-binding site. Our studies extend structural analyses of MEF2 and provide additional structural information to guide further functional studies of MEF2B inside cell. Results Overall structure of Apo MEF2B We determined the crystal structure of the MADSbox/MEF2 domain of MEF2B (residue 1-93) at 2.3 Å resolution (Table S1). There are three MEF2B Apo dimers in the crystal asymmetric unit (Figure 1A). Two MEF2B Apo dimers (dimer 1 and dimer 2) adopt the classical layered, sandwich-like structures as previously observed: helix H1 from both monomers forming the bottom layer, strand S1-S3 from both monomers forming the middle layer, and helix H2 from both monomers forming the top layer. Surprisingly, while the third MEF2B Apo dimer (dimer 3) adopts the overall three-layered sandwich structure, its C terminal helix flips up into the MEF2 cofactor interacting groove. Structure comparison between the 3 Apo MEF2B dimers in the asymmetric unit and between Apo MEF2B with previously reported structures of MEF2B in the MEF2B/Cabin1/DNA and MEF2B/HDAC9/DNA complexes shows a high overall structural similarity (RMSD between 0.16 to 0.54 Å for Ca atoms alignment. Figure 1). The conformation of DNA interacting residues in Apo MEF2B and the conformation of helix H3 in Apo MEF2B dimer 3 are analyzed in detail below.

Figure 1. Overall structure of Apo MEF2B. (A) Crystal asymmetric unit. The three MEF2B dimers are colored in blue, green, and red. This color scheme is followed in the whole report. (B) Structure comparison between the three MEF2B dimers in crystal asymmetric unit and MEF2B reported in literature: MEF2B/Cabin1/DNA complex (PDB 1N6J) and MEF2B/HDAC9/DNA complex (PDB 1TQE).

DNA-binding by the N-terminal loop and helix H1 of Apo MEF2B Our structure provides an opportunity to compare the DNA binding interface of MEF2 before and after DNAbinding. There is no electron density for the first 5-8 amino acid residues (MGRKKIQI) in all three dimers of MEF2B Apo, whereas in DNA-bound MEF2B complexes these residues have well-defined electron density. These residues contact DNA in the minor groove and are important in DNA binding and recognition 12,20,21. In addition, the visible portion of the N-terminal loop in our MEF2B Apo structures adopts various conformations among different Apo dimers. This part of the N terminal loop in MEF2B/DNA complexes has similar conformation in various complexes (Figure S2), which are different from that seen in the apo MEF2 structures. These observations suggest that without DNA binding, the MEF2B Nterminal loop is highly flexible. The amino acids sequence of the DNA binding helix H1 of MEF2 is the same among the four family members (MEF2 A-D). Helix H1 is important for DNA binding. K23, K30, which are key DNA interacting residues in the major groove, and R24, K31, which are key DNA interacting residues in the minor groove, are all located in this helix (Figure 2) 8,12,22,23. We compared the DNA binding helix H1 between Apo MEF2B and high resolution (2.4 Å or higher) MEF2 complexes (MEF2B/Cabin1/DNA and MEF2A/DNA) in literature 8,12 . The overall conformation of helix H1 and the spacing between helix H1 from the two monomers in a MEF2 dimer are highly conserved (RMSD between 0.10 Å to 0.29 Å for Ca atom alignment) (Figure 3). We

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Biochemistry

observed clear electron density (2Fo-Fc map at 1 sigma level) of key DNA interaction residues on helix H1 (K23, R24, K30, K31, and E34) in all the Apo MEF2B structures except for K23, K30 and K31, whose sidechains only showed partial electron density on one monomer of the Apo MEF2B dimer 3. The structural comparison shows that the side chain conformations of R24 and E34 are the same in Apo MEF2B and in MEF2B/DNA or MEF2A/DNA complexes, while different rotamers are observed for K23, K30, and K31 side chains in Apo MEF2B and between Apo MEF2B and MEF2B/DNA or MEF2A/DNA complexes (Figure 3). These detailed analyses show that even without DNA binding, the key DNA interacting residues on MEF2B, especially R24 and E34, are ordered, and stabilized by forming salt bridge between each other. This result shows that the main DNA-binding interface formed by helix H1 is largely preformed, while the N terminal loops undergo binding-induced folding.

Figure 2. Key DNA interacting residues on MEF2B helix 1. (A) DNA is shown in surface mode. Helix 1 is shown in cartoon and key residues are shown as sticks (K23, R24, K30, and K31). Model is adapted from MEF2B/Cabin1/DNA complex (PDB 1N6J). (B) Zoom-in view of key minor groove interacting residues (R24 and K31) and key residue pairs (R24 and E34)

Figure 3. Comparison of key DNA interacting residues on MEF2 helix 1. (A) Structural alignment of helix 1 between three MEF2B dimers in Apo MEF2 structure (blue, green, and red) and helix 1 in MEF2A/DNA complex (yellow) and MEF2B/Cabin1/DNA complex (grey). DNA is shown in surface mode. Helix 1 is shown in ribbon mode and key interacting residues are shown in sticks.

(B) zoom-in view of key DNA interacting residues on helix 1 showing that the conformation of R24 and E34 is the same while the sidechain rotamer conformation of residues K30 and K31 vary among different structures. Orange dots between E34 and R24 indicate the hydrogen bond between these two residues.

Intramolecular binding of the ligand interaction groove by the C-terminal helix H3 Crystal structures of MEF2/co-factor complexes show that MEF2 cofactors (cabin 1, HDAC9, p300, and FAK) use an amphipathic helix to bind the hydrophobic groove between helix H2 on the top of the MEF2 sandwich structure 8,11,15,24. The hydrophobic face of the amphipathic helices from various cofactors share conserved features and binding mechanisms to MEF2. For example, in the MEF2B/Cabin1/DNA complex crystal structure, Ile 2164, Thr 2168, Ile 2176 and Leu 2177 from the Cabin1 amphipathic helix interact with MEF2B Leu66 and Leu67 in the hydrophobic groove through van der Waals contacts (Figure 4) 8. When solving the crystal structure, we used the MEF2B structure from the published MEF2B/DNA complex as a partial search model in molecular replacement (MR). Even though helix H3 in the search model is pointing toward the bottom of the MEF2 sandwich structure, we observed strong positive helical electron density in the hydrophobic ligand binding groove of Apo MEF2B dimer 3, whereas no electron density was visible in the original location of helix H3. These crystallographic analyses indicate that helix H3 of Apo MEF2B dimer 3 has a very different conformation from all the MEF2 structures reported so far, namely, helix H3 flipped up to mimic the amphipathic helix of MEF2 co-factors to bind the hydrophobic groove between helix 2 (Figure 4). Because there are two possible alternative conformations of helix H3 resulting from helix H3 flipping from either MEF2B monomer; electron density of this region is the superposition of helix H3 in both orientations. The electron density indicated one of the orientations is more dominant. As a result, we are able to build one major conformation model into the electron density, which is supported by subsequent composite omit map (Figure S3). Detailed analyses of helix H3 show that hydrophobic residues I84, L88 interact with the MEF2 ligand-binding groove, while hydrophilic residues T82 and E86 are facing solvent (Figure 4). Structural-based sequence alignment shows that helix H3 from other family members (MEF2A, C, D) has similar sequence pattern as MEF2B, all of which share key interacting residues with MEF2 binding helices from cofactors (Figure S3) 15.

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aggressive cutaneous squamous cell carcinoma, Merkel cell carcinoma, and gastric adenocarcinoma patients respectively 29–31. Based on our structure, the E34K or E34A mutations are likely to disrupt R24 conformation; The consequence could lead to non-specific DNA interaction or loss of DNA interaction of MEF2.

Figure 4. Helix H3 in Apo MEF2B dimer 3 mimics MEF2 cofactor in binding to the MEF2 hydrophobic groove. (A) Detailed interactions between Cabin1 amphipathic helix (green) to MEF2 hydrophobic groove (grey). Residues of interests are shown as sticks. Cabin1 residues are labeled in red. (B) Detailed interactions between helix H3, which is also amphipathic, to MEF2 hydrophobic groove in Apo MEF2B dimer 3. Residues of interests in helix H3 are shown as sticks in green and residues of interests in helix H2 are shown as sticks in grey.

Discussion We determined the crystal structure of MEF2B Apo protein. Our structure shows that MEF2B adopts a largely preformed DNA binding conformation prior to binding to DNA; namely, the spacing between the DNA binding helix H1 is preformed in the apo state and several key DNA interacting residues on H1 (R24 and E34) retain similar rotamer conformations before and after DNA binding. Our structure also shows that the Cterminal helix H3 on the MEF2-specific domain could flip up onto the hydrophobic ligand binding groove, suggesting a potential self-regulatory role of this region. Since MEF2 proteins have conserved DNA binding and MEF2-specific domains, these insights from our study of Apo MEF2B structure may apply to other MEF2 family members. MEF2 belongs to the MADS box superfamily of proteins, which are well known for binding DNA through shape recognition 21–23. Our structure shows that DNA binding does not induce significant structure change in MEF2 DNA binding helix H1 but induces the N-terminal loop to become ordered. Based on these observations, we speculate that MEF2 may first locate its target sites primarily via shape recognition by helix H1, followed by specific interactions of the N- terminal loop in the major and minor grooves. Our structural analyses also reveal a highly conserved feature of MEF2, namely, E34 stabilizes the side chain conformation of a major DNA interacting residue R24, suggesting an important role of E34 in DNA binding. The R24 and E34 pair are evolutionarily conserved in MADS box transcription factors across fungi, plants and animals 25–28. Additionally, MEF2A E34K, MEF2D E34K, and MEF2D E34A point mutations are found in

We observed that helix H3 on the MEF2-specific domain could flip up to bind the MEF2 ligand binding groove, in a manner similar to the amphipathic helices of MEF2 cofactors. We noticed that in all three dimers of MEF2B in the asymmetric unit, helix H3 only flipped up in dimer 3 but not in dimer 1 and dimer 2. We analyzed the crystal packing of our structure and found that the cofactor binding groove in dimer 1 and dimer 2 are blocked by part of the N terminal loop from crystal symmetry mates (Figure S4). The packing analysis also suggests that helix H3 in dimer 3 is likely attracted to the cofactor binding groove by positive interactions, which is also consistent with the sequence similarity between H3 and the MEF2-binding helices from various cofactors. While the physiological significance of this interaction requires further study, the occupancy of helix H3 in the ligand binding groove would prevent the recruitment of MEF2 cofactors (HDAC, Cabin1, p300, FAK etc). We therefore propose that helix H3 may have a self-regulatory role in MEF2 function. Because helix H3 is frequently targeted by cancer mutations and posttranslational modification (PTM) 2,9,10,32, it is also tempting to speculate that these mutations and PTM could perturb the conformation of helix H3 and therefore the function of MEF232. Furthermore, we noticed that in dimer 3 the loop between helix H2 and strand S3 (H2-S3 loop) and the strand S3 have no electron density. The flexibility of this region was indicated by molecular dynamic studies previously and may be important for helix H3’s ability to flip into the ligand binding groove33. Early X-ray and NMR studies suggest strand S3 and helix H3 were disordered 12,13. Our study shows that helix H3 could be in two positions: pointing to DNA or flipped into the ligand binding groove. These two conformations could switch fast in solution, and this may be one interpretation of the disordered nature observed in NMR studies. In the cellular context, the conformation of helix H3 flipped up into the MEF2 cofactor groove could be stabilized by other yet unknown cofactors. Helix H3 has a similar amino acid sequence pattern as the consensus of MEF2-binding helices of MEF2 cofactors; in addition, helix H3 interacts with the ligand binding groove in a similar way as MEF2 cofactors do. The flipping up of helix H3 could reorient the C-terminal transcription activation domain and therefore potentially regulate MEF2’s function under certain cellular conditions.

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Biochemistry

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Experimental Procedures, Tables S1, and Figures S1−S4 (PDF)

AUTHOR INFORMATION Corresponding Author

Email: [email protected] Author Contributions

The manuscript was written through contributions of all authors. Funding Sources

No competing financial interests have been declared. This work was supported by National Institute of Health Grants R01AI113009 and U54DK107981.

ACKNOWLEDGMENT

(5) Pasqualucci, L., Trifonov, V., Fabbri, G., Ma, J., Rossi, D., Chiarenza, A., Wells, V. A., Grunn, A., Messina, M., Elliot, O., Chan, J., Bhagat, G., Chadburn, A., Gaidano, G., Mullighan, C. G., Rabadan, R., and Dalla-Favera, R. (2011) Analysis of the coding genome of diffuse large B-cell lymphoma. Nat. Genet. 43, 830–837. (6) Lohr, J. G., Stojanov, P., Lawrence, M. S., Auclair, D., Chapuy, B., Sougnez, C., Cruz-Gordillo, P., Knoechel, B., Asmann, Y. W., Slager, S. L., Novak, A. J., Dogan, A., Ansell, S. M., Link, B. K., Zou, L., Gould, J., Saksena, G., Stransky, N., Rangel-Escareno, C., Fernandez-Lopez, J. C., Hidalgo-Miranda, A., Melendez-Zajgla, J., Hernandez-Lemus, E., Schwarz-Cruz y Celis, A., Imaz-Rosshandler, I., Ojesina, A. I., Jung, J., Pedamallu, C. S., Lander, E. S., Habermann, T. M., Cerhan, J. R., Shipp, M. A., Getz, G., and Golub, T. R. (2012) Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc. Natl. Acad. Sci. U. S. A. 109, 3879–3884. (7) Molkentin, J. D., and Olson, E. N. (1996) Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors. Proc. Natl. Acad. Sci. U. S. A. 93, 9366–73. (8) Han, A., Pan, F., Stroud, J. C., Youn, H. D., Liu, J. O., and Chen, L. (2003) Sequence-specific recruitment of transcriptional corepressor Cabin1 by myocyte enhancer factor-2. Nature 422, 730– 734. (9) Ying, C. Y., Dominguez-Sola, D., Fabi, M., Lorenz, I. C., Hussein, S., Bansal, M., Califano, A., Pasqualucci, L., Basso, K., and Dalla-Favera, R. (2013) MEF2B mutations lead to deregulated expression of the oncogene BCL6 in diffuse large B cell lymphoma. Nat. Immunol. 14, 1084–1092.

The authors thank Satyanarayan Rao, Dr. John Petruska, Dr. Ana Carolina Dantas Machado and Dr. Remo Rohs for helpful discussion. The authors thank staff and scientists from Advanced Light Source beamline 8.2.1 for their help with X-ray diffraction data collection. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract DE-AC02-05CH11231.

(10) Pon, J. R., Wong, J., Saberi, S., Alder, O., Moksa, M., Grace Cheng, S. W., Morin, G. B., Hoodless, P. A., Hirst, M., and Marra, M. A. (2015) MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation. Nat. Commun. 6, 7953.

ABBREVIATIONS

(12) Santelli, E., and Richmond, T. J. (2000) Crystal structure of MEF2A core bound to DNA at 1.5 A resolution. J. Mol. Biol. 297, 437–449. (13) Huang, K., Louis, J. M., Donaldson, L., Lim, F. L., Sharrocks, A. D., and Clore, G. M. (2000) Solution structure of the MEF2A-DNA complex: structural basis for the modulation of DNA bending and specificity by MADS-box transcription factors. EMBO J. 19, 2615– 28. (14) Wu, Y., Dey, R., Han, A., Jayathilaka, N., Philips, M., Ye, J., and Chen, L. (2010) Structure of the MADS-box/MEF2 domain of MEF2A bound to DNA and its implication for myocardin recruitment. J. Mol. Biol. 397, 520–533. (15) Han, A., He, J., Wu, Y., Liu, J. O., and Chen, L. (2005) Mechanism of recruitment of class II histone deacetylases by myocyte enhancer factor-2. J. Mol. Biol. 345, 91–102. (16) De Angelis, L., Borghi, S., Melchionna, R., Berghella, L., Baccarani-Contri, M., Parise, F., Ferrari, S., and Cossu, G. (1998) Inhibition of myogenesis by transforming growth factor beta is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm. Proc. Natl. Acad. Sci. U. S. A. 95, 12358–63. (17) Chen, S. L., Wang, S. C., Hosking, B., and Muscat, G. E. (2001) Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells. Mol. Endocrinol. 15, 783–796. (18) Neely, M. D., Robert, E. M., Baucum, A. J., Colbran, R. J., Muly, E. C., and Deutch, A. Y. (2009) Localization of myocyte enhancer factor 2 in the rodent forebrain: regionally-specific cytoplasmic expression of MEF2A. Brain Res. 1274, 55–65.

MEF2: Myocyte enhancer factor 2 FAK: Focal adhesion kinase MR: Molecular Replacement Cabin1: Calcineurin-binding protein cabin-1 p300: Histone acetyltransferase

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