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Received: 21 December 2016 Accepted: 3 May 2017 Published: xx xx xxxx

Structural Characterization of the SMRT Corepressor Interacting with Histone Deacetylase 7 Danielle C. Desravines1,2, Itziar Serna Martin1,2,4, Robert Schneider   3,5, Philippe J. Mas1,2, Nataliia Aleksandrova1,2, Malene Ringkjøbing Jensen3, Martin Blackledge3 & Darren J. Hart   1,2 The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple transcription factors and chromatin modifying enzymes. Here we have studied its interaction with HDAC7, a class IIa deacetylase that interacts with the corepressor complex together with the highly active class I deacetylase HDAC3. The binding site of class IIa deacetylases was previously mapped to an approximate 500 amino acid region of SMRT, with recent implication of short glycine-serine-isoleucine (GSI) containing motifs. In order to characterize the interaction in detail, we applied a random library screening approach within this region and obtained a range of stable, soluble SMRT fragments. In agreement with an absence of predicted structural domains, these were characterized as intrinsically disordered by NMR spectroscopy. We identified one of them, comprising residues 1255–1452, as interacting with HDAC7 with micromolar affinity. The binding site was mapped in detail by NMR and confirmed by truncation and alanine mutagenesis. Complementing this with mutational analysis of HDAC7, we show that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix. The major role of histone deacetylases (HDACs) is to deacetylate lysines of different protein substrates, commonly histones, where they lead to compaction of the chromatin structure and gene repression. The human HDAC family comprises 18 members divided into 4 classes based on sequence homology. Among these are class I HDACs 1, 2, 3 and 8 with a catalytic domain of 377–488 amino acids1, 2, and class II HDACs that are 855 to 1122 amino acids long and contain an additional N-terminal regulatory domain1–3. Class II is further subdivided into IIa (HDAC4, 5, 7 and 9) and IIb (HDAC6 and 10)1, 4. Crystal structures of class I and II catalytic domains reveal a common active site with a deep pocket containing a conserved zinc-binding site. HDAC7, first identified as a component of a multiprotein complex5, has a similar catalytic domain structure to other HDACs6–8. As with other class IIa enzymes, the mechanistically important tyrosine (Tyr306) is replaced by a histidine, resulting in a barely detectable or “cryptic” enzymatic activity6, 7, 9. This inactivation of an otherwise conserved active site suggests that class IIa enzymes may instead function as acetyl-lysine readers similar to bromodomains10, with deacetylation activity being contributed by class I HDACs in the context of a corepressor complex. HDAC7 functionality is dependent on the presence of HDAC3, and both enzymes were shown to associate with SMRT and N-CoR1 corepressors11. SMRT (silencing mediator of retinoid acid and thyroid hormone receptor), also known as N-CoR2, is a 2525 amino acid protein identified as binding to unliganded nuclear receptors12 (Fig. 1). It has 40% sequence identity to N-CoR1; however, the two proteins are non-redundant and knockout of either of them results in embryonic death in mice13, 14. Deregulation of corepressor function via defective protein-SMRT interactions has been implicated in many cancers15 including acute promyelocytic16, 17, acute myeloid18 and common acute lymphoblastic leukemias19, as well as other conditions, e.g. resistance to thyroid hormone (RTH) syndrome20.

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European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS90181, 38042, Grenoble Cedex 9, France. 2Unit of Virus Host-Cell Interactions (UVHCI), University Grenoble Alpes, CNRS, EMBL, 38042, Grenoble, France. 3Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, 38044, Grenoble, France. 4Present address: Dunn School of Pathology, University of Oxford, South Parks Road, OX13 RE, Oxford, UK. 5 Present address: University Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France. Danielle C. Desravines, Itziar Serna Martin and Robert Schneider contributed equally to this work. Correspondence and requests for materials should be addressed to R.S. (email: [email protected]) or D.J.H. (email: [email protected]) Scientific Reports | 7: 3678 | DOI:10.1038/s41598-017-03718-5

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Figure 1.  Functional and structural domains of the SMRT protein. Top, general schematic of the entire protein; bottom, close-up view of the region investigated in this work.

Despite its large size, only two small structured SANT-like domains have been characterized in SMRT. One is the deacetylase activating domain (DAD) that recruits and activates HDAC3 in a inositol phosphate-mediated fashion21, 22, the other forms a histone interaction domain23. HDAC7 has been shown to bind within a 500 amino acid region of SMRT (residues 1289–1793)24 via a second zinc ion on the surface of the catalytic domain7. Analysis of the SMRT sequence using computational predictors suggests that most of its chain is unstructured (Fig. S1) and that, consequently, it belongs to the class of intrinsically disordered proteins (IDPs) that are functional despite a lack of stable three-dimensional structure. Research over the last two decades has underlined the important roles of IDPs in biology, notably in processes such as signaling, regulation, or mediation of protein-protein interactions25–31. SMRT conforms well to this profile, forming interactions with a wide range of proteins, including HDACs, nuclear receptors and transcription factors18, 32–34. Development of class IIa HDAC inhibitors (HDIs) is complicated by the very low catalytic activity of these enzymes and a poor understanding of their function35. However, class IIa HDACs have a number of roles in development and physiology36 and are implicated in cancer15, 37, 38, making them attractive pharmacological targets. Most inhibition strategies target the HDAC active site with metal-binding compounds (e.g. trichostatin A or suberanilohydroxamic acid). Generally, HDIs lack specificity due to cross-reactivity with other HDACs and metalloenzymes39, although there has been some progress towards class IIa-specific HDIs39. An alternative strategy to inhibit class IIa HDACs would be to block their recruitment into the SMRT corepressor complex, which might avoid problems of pan-HDAC or metalloenzyme binding. Towards this goal, we screened a library of SMRT constructs to obtain soluble fragments, then used NMR spectroscopy and biophysical methods to localize and characterize the HDAC7-SMRT binding interface. NMR is well suited for studying intrinsically disordered proteins40 and allowed us to characterize both structural propensities and the HDAC7 binding site of SMRT with single-residue precision. Our data lay the foundation for the development of selective inhibitors of corepressor assembly.

Results

Expression of soluble fragments of SMRT.  The region of SMRT interacting with class IIa HDACs was previously localized to the region of repression domain (RD) 3 using yeast two-hybrid deletion analysis11, 41, 42 and more specifically to residues 1289–179324 (Fig. 1). We synthesized an E. coli codon-optimized SMRT gene fragment (encoding residues 1089–1993) and screened it for soluble constructs using the ESPRIT (expression of soluble proteins by random incremental truncation) method43, 44. Initially we obtained 51 constructs yielding soluble purifiable protein visible by western blot. Most fall in the size range of 100 to 200 amino acids and, collectively, cover most of the SMRT fragment investigated. Based on yield from small-scale expression trials, proteolytic stability and sequence position, six constructs (1122–1254, 1255–1452, 1276–1405, 1784–1993, 1798–1966 and 1882–1993) were selected for isotope-labeled expression, purification and NMR spectroscopy. In agreement with bioinformatic predictions (e.g. IUPRED45), resistance to aggregation at 80 °C and abnormal migration of proteins on SDS-PAGE gels, the reduced dispersion of signals in the 1H dimension of 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectra confirmed the protein fragments as disordered (Supplementary Fig. S2). Identification and characterization of HDAC7-interacting SMRT fragments.  For interaction studies, equimolar mixtures of 15N-labeled SMRT constructs and unlabeled HDAC7 protein were prepared. SMRT (1255–1452) and SMRT (1276–1405) interact with HDAC7, as apparent from reduced or vanishing spectral peak intensities (Fig. 2). Due to its better long-term stability in NMR experiments, SMRT (1255–1452) was selected for subsequent studies. The interaction of HDAC7 and SMRT (1255–1452) was measured by surface plasmon resonance (SPR) (Fig. 3a). The data could not be analyzed using a simple 1:1 binding model; however, a high quality global fit was obtained using a two-step (three-state; A + B   AB(initial)  AB(final)) reaction model, consistent with the interaction occurring via an initial non-specific encounter complex and/or a folding-upon-binding mechanism as described for other IDPs46, 47. The overall Kd of the interaction was measured as 2.1 µM (Table S1). Using microscale thermophoresis (MST)48 as a complementary solution-based method, we measured a Kd of 2.8 µM for SMRT(1255–1452)-HDAC7 binding (Fig. 3b). Isothermal titration calorimetry (ITC) measurements yielded a Scientific Reports | 7: 3678 | DOI:10.1038/s41598-017-03718-5

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Figure 2.  Interaction of SMRT fragments with HDAC7. (a,b) Superposition of 15N-1H HSQC spectra of SMRT fragments 1255–1452 (a) and 1784–1993 (b) in the absence (red) and presence (blue) of equimolar amounts of unlabeled HDAC7. In (a), selected peaks exhibiting reduced intensity in the presence of HDAC7 are indicated by arrows. See also Supplementary Fig. S4. (c) Relative peak intensity ratios I/I0 between spectra with and without added HDAC7 of SMRT(1255–1452) (red) and SMRT(1784–1993) (black), showing significant signal attenuation only for SMRT(1255–1452). Since sequential resonance assignment was not performed for SMRT(1784–1993), peaks are arbitrarily numbered and sorted for relative intensity ratio for both SMRT fragments to facilitate comparison. Only non-overlapping peaks were considered. Additional peaks for the SMRT(1784–1993) construct result from the presence of a C-terminal biotin tag.

slightly higher Kd of 14.1 µM, which can be rationalized by the visible precipitation of HDAC7 which hampered ITC experiments at higher concentrations. Taken together, our data thus situate the Kd of the interaction in the 2–3 µM range. Based on this Kd value, we measured HDAC7 deacetylase activity in the presence of SMRT (1255–1452) at a concentration where most HDAC7 would be bound ([HDAC7]:[SMRT(1255–1452)] = 8 nM:10 µM) in order to test whether the reported cryptic enzymatic activity on acetyllysine substrates6 could be enhanced. Neither SMRT(1255–1452) nor any of the other purified fragments increased the activity of HDAC7 on acetyllysine or trifluoroacetyllysine substrates (Supplementary Fig. S3). Similar negative results have been reported for other SMRT peptides49, consistent with the view that SMRT-mediated deacetylation in vivo is performed by HDAC3.

Structural characterization of SMRT(1255–1452).  We obtained NMR resonance assignments for 97% of the assignable sequence of SMRT(1255–1452) using standard BEST-type triple-resonance experiments50 on a 13C,15N-labeled sample. We used the resultant chemical shifts and transverse 15N relaxation rates (R2) and {1H}-15N heteronuclear nuclear Overhauser enhancements (hetNOEs) to identify regions of transient secondary structure (Fig. 4). Secondary structure propensity (SSP)51 calculated from chemical shifts exhibits elevated positive values in four continuous stretches of SMRT(1255–1452), namely residues 1320–1324, 1334–1338, Scientific Reports | 7: 3678 | DOI:10.1038/s41598-017-03718-5

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Figure 3.  Determination of SMRT(1255–1452)–HDAC7 binding affinity. (a) Surface plasmon resonance curves of 8, 6, 2, 1, and 0.5 µM HDAC7 (upper to lower curve) binding to immobilized SMRT(1255–1452), yielding a Kd of 2.1 µM. Dashed curves represent fits to the data (solid lines) using a three-state (two-step) binding model. (b) Microscale thermophoresis data of HDAC7 binding to fluorescently labeled SMRT. The solid line represents a fit to the data yielding a Kd of 2.8 µM. Vertical axis shows the ratio of fluorescence (F) measured with and without laser heating.

Figure 4.  NMR analysis of SMRT(1255–1452). (a) Secondary structure propensity (SSP) values based on experimental Cα and Cβ chemical shifts. Positive values indicate α-helical, negative values β-extended propensity. Cutoff values of 0.1 are indicated by horizontal dashed lines. (b) 1H-15N heteronuclear nuclear Overhauser enhancement (hetNOE) values at 25 °C and 600 MHz 1H Larmor frequency. (c) Transverse (R2) 15N relaxation rates measured at 25 °C and 600 MHz 1H Larmor frequency. Regions with SSP values above 0.1 and elevated hetNOE values, indicative of transient helical structure, are indicated with gray shading. Error bars in panels (b) and (c) represent parameter standard deviations estimated based on noise levels in NMR spectra. Residues without data in panels (b) and (c) are prolines (which do not appear in HSQC-type spectra), have overlapping resonance signals, or are unassigned due to insufficient signal intensity. Scientific Reports | 7: 3678 | DOI:10.1038/s41598-017-03718-5

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Figure 5.  Interaction of SMRT(1255–1452) with HDAC7. Bar plots of relative signal intensities (HDAC7complexed vs. free) for SMRT(1255–1452) residues in HSQC spectra with different amounts of added wtHDAC7 (see Supplementary Fig. S4). The longest region of transient helical structure in free SMRT(1255–1452) (residues 1370–1386) is indicated by gray shading. Missing bars correspond to proline residues, residues whose HSQC peaks overlap, or unassigned residues. 1370–1386 and 1400–1407, indicating transient helical structure (Fig. 4a). HetNOE values are also elevated in these regions, consistent with reduced mobility on the pico- to nanosecond timescale due to transient structure formation (Fig. 4b). For residues 1370–1386 whose SSP values are particularly large, a sizable increase of transverse 15N relaxation rates, with a periodicity of 3 to 4 residues, is also apparent (Fig. 4c), further supporting the existence of helical structure propensity in this region. Notably, all four transiently helical regions are preceded by Asp or Ser residues, which can N-cap α-helices by accepting hydrogen bonds from free amides via their sidechains52, 53. Several examples of transient helices in disordered proteins that are initiated by N-capping residues have been described54–56, suggesting that the presence of transient helical structure in SMRT is encoded in its primary sequence.

Identification of the HDAC7 binding site of SMRT by NMR, mutagenesis and truncation.  We

used SMRT(1255–1452) resonance assignments to identify the HDAC7 binding site. SMRT(1255–1452) was titrated with increasing amounts of unlabeled HDAC7, and the attenuation of peak intensities in HSQC spectra was monitored. We observed a clear and systematic decrease in intensity in a large region in the center of the molecule (residues 1310 to 1430), with the strongest attenuations observed in residues 1360–1387 (Fig. 5, Supplementary Fig. S4). These data suggest that the region of residues 1370–1386, containing the most pronounced pre-formed transient helical structure, binds HDAC7 and becomes extended to a longer helix, again consistent with a folding-upon-binding interaction46, 57. The actual binding most likely occurs in the strongly attenuated residues 1360–1387, contained in one of the predicted protein interaction sites of SMRT (Supplementary Fig. S1). We investigated this hypothesis by fitting the dependence of SMRT(1255–1452) peak intensities on the HDAC7 concentration to obtain an estimate of the Kd. While this method does not allow for a precise Kd determination, it yields micromolar values approaching those obtained from SPR, MST and ITC for residues 1360–1386 (

Structural Characterization of the SMRT Corepressor Interacting with Histone Deacetylase 7.

The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple tr...
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