Molecular Basis for Recognition of Methylated and Specific DNA

Molecular basis for recognition of methylated and speciﬁc DNA sequences by the zinc ﬁnger protein Kaiso

Bethany A. Buck-Koehntopa, Robyn L. Stanﬁelda, Damian C. Ekierta, Maria A. Martinez-Yamouta, H. Jane Dysona, Ian A. Wilsona,b, and Peter E. Wrighta,b,1

aDepartment of Molecular Biology and bSkaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037

Contributed by Peter E. Wright, August 7, 2012 (sent for review July 19, 2012) Methylation of CpG dinucleotides in DNA is a common epigenetic in cancer cells (14). Specific Kaiso-binding sites are found in the modification in eukaryotes that plays a central role in maintenance promoters of several target genes (matrilysin, cyclin-D1, siamois, of genome stability, gene silencing, genomic imprinting, develop- c-myc,andWnt11) regulated by the Wnt signaling pathway, ment, and disease. Kaiso, a bifunctional Cys2His2 zinc finger protein which plays a critical role in early development and tumor implicated in tumor-cell proliferation, binds to both methylated CpG progression (9, 15, 16). The cytoplasmic armadillo repeat pro- (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) tein p120 catenin (p120ctn) binds Kaiso and relieves Kaiso- and represses transcription by recruiting chromatin remodeling co- mediated transcriptional repression (6, 17, 18). The interaction repression machinery to target genes. Here we report structures of of Kaiso with p120ctn has been implicated in regulation of ca- the Kaiso zinc finger DNA-binding domain in complex with its non- nonical and noncanonical Wnt signaling pathways through methylated, sequence-specific DNA target (KBS) and with a symmet- a mechanism that involves KBS recognition (15, 16). Addi- rically methylated DNA sequence derived from the promoter region tionally, there is increasing evidence that Kaiso expression lev- of E-cadherin. Recognition of specific bases in the major groove of els are elevated in tumorigenesis, indicating that Kaiso may play the core KBS and mCpG sites is accomplished through both classical a direct role in modulating cancer through transcriptional and methyl CH···O hydrogen-bonding interactions with residues in control of aberrantly methylated and KBS-containing genes (14, the first two zinc fingers, whereas residues in the C-terminal exten- 19, 20). It still is unclear whether the transcriptional regulation sion following the third zinc finger bind in the opposing minor of the two distinctly different DNA sequences is synergistic in groove and are required for high-affinity binding. The C-terminal nature or mutually exclusive. region is disordered in the free protein and adopts an ordered struc- Although structures of the SRA (UHRF1) (21–23) and MBD ture upon binding to DNA. The structures of these Kaiso complexes (MBD1, MeCP2, and MBD2) (24–26) domains in complex with provide insights into the mechanism by which a zinc finger protein methylated DNA have been determined, the structural basis for can recognize mCpG sites as well as a specific, nonmethylated reg- recognition of both mCpG and KBS sites by the Kaiso family is ulatory DNA sequence. unknown. Here we report crystal and solution structures of the Kaiso zinc finger domain in complex with its nonmethylated, se- protein–DNA interaction | NMR spectroscopy | X-ray crystallography | quence-specific DNA site (KBS) (Fig. 1A) and the crystal struc- folding upon binding | intrinsic disorder ture of the complex with an oligonucleotide derived from the promoter region of E-cadherin, a known Kaiso-binding site (7, 27) n eukaryotes, DNA methylation is a common epigenetic mod- that contains two sequential, symmetrically methylated CpG sites Iification that is central to the maintenance of genome stability, (MeECad) (Fig. 1B). In normal cells, E-cadherin is an important gene silencing, genomic imprinting, development, and disease mediator of cell–cell adhesion, but hypermethylation of the pro- (1, 2). Methyl-CpG–binding proteins (MBPs) mediate these promoter in several cancers results in down-regulation of E-cadherin cesses by binding to methylated DNA signals and recruiting expression and has been correlated with increasing invasion and chromatin remodeling corepressor complexes, resulting in com- metastasis (28). The current studies provide insights into the paction of chromatin into its transcriptionally inactive state (3). mechanism by which Kaiso recognizes both methylated DNA and To date, three classes of MBPs that recognize 5-methyl cytosine the unmethylated KBS DNA sequence. Remarkably, the molec- (5mC) have been identified. The SRA domain family has speci- ular interactions that mediate binding to these distinct DNA ficity for hemimethylated sites and is required for maintenance target sites are very similar. methylation during DNA replication (4). In contrast, the methyl CpG-binding domain (MBD) and Kaiso families of MBPs function Results and Discussion as essential mediators of epigenetically controlled gene silencing Overall Structure of the Kaiso:DNA Complexes. The crystal structure by recognizing symmetrically methylated CpG sites (5). Kaiso is a of the Kaiso:KBS complex was determined at 2.4-Å resolution MBP belonging to the BTB/POZ (broad complex, tramtrak, bric à using single-wavelength anomalous dispersion from the zinc, and brac/pox virus and zinc finger) subfamily of transcription factors that the Kaiso:MeECad complex then was solved by molecular re- recognize cognate DNA sequences through a C-terminal zinc finger placement at 2.8-Å resolution as detailed in SI Methods. The domain; the N-terminal BTB/POZ domain mediates protein–protein interactions (6). Kaiso is a POZ protein that participates in both methyl-dependent and sequence-specific transcriptional re- Author contributions: B.A.B.-K., M.A.M.-Y. and P.E.W. designed research; B.A.B.-K., R.L.S., fi D.C.E., and M.A.M.-Y. performed research; B.A.B.-K., R.L.S., D.C.E., H.J.D., I.A.W., and P.E.W. pression, using its three Cys2His2 zinc ngers to recognize either two BIOCHEMISTRY consecutive symmetrically methylated CpG dinucleotides (mCpG) analyzed data; and B.A.B.-K., H.J.D., I.A.W., and P.E.W. wrote the paper. (7,8)oraTCCTGCNAconsensus(termed“KBS”) (9). Although The authors declare no conflict of interest. originally it was reported that Kaiso only requires zinc fingers 2 and Data deposition: Atomic coordinates and structure factors have been deposited in Protein 3 for DNA recognition (9), we determined that Kaiso in fact Data Bank, www.pdb.org [PDB ID codes 4F6M (crystal structure of the KBS complex), 4F6N fi (crystal structure of the MeECad complex), and 2LT7 (NMR structures)] and resonance requires all three zinc ngers plus adjacent regions for structural assignments in the Biological Magnetic Resonance Bank, http://www.bmrb.wisc.edu stability and high-affinity binding to both sequence classes (10). [accession no. 18462 (NMR assignments)]. Transcriptional repression by Kaiso has implications in both 1To whom correspondence should be addressed. E-mail: [email protected]. – development and cancer (11 13). Kaiso binds to and silences This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. aberrantly methylated tumor-suppressor and DNA-repair genes 1073/pnas.1213726109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1213726109 PNAS | September 18, 2012 | vol. 109 | no. 38 | 15229–15234 Downloaded by guest on September 27, 2021 heteronuclear single-quantum coherence (HSQC) spectrum of free Kaiso but undergo large shifts in the spectrum of the KBS complex, indicating formation of folded structure in the presence of DNA (Fig. 2A). Backbone {1H}-15N heteronuclear nuclear Overhauser effect (NOE) measurements (Fig. 2B)conﬁrm that this region is highly ﬂexible in the free protein but becomes ordered, with NOE values similar to those of the rest of the protein, in the presence of KBS. The structure induced in the C-terminal region is stabilized by incorporation into the ZF3 β-sheet, by hydrophobic packing and hydrogen-bonding interactions with ZF2, and by interactions with the phosphate backbone (Figs. S1 and S3A).

Molecular Determinants of KBS and MeECad DNA Recognition. In both the KBS and MeECad complexes, the zinc ﬁngers are anchored to the DNA by an extensive network of direct and water- mediated hydrogen bonds to the phosphate backbone as well as by van der Waals interactions with the sugar rings (Fig. 3A). Subtle differences are observed in the phosphate backbone interactions for the KBS and MeECad complexes (Fig. S2). ZF3 is anchored ﬁrmly to the DNA backbone by a network of hydrogen bonds from a cluster of tyrosine residues (Fig. 3B). Residues 590–594 form a structured loop, which is anchored to the side chain of Glu-547 in the ZF2–ZF3 linker by hydrogen bonds involving Arg-590 and Ser-591 backbone amides, and packs, by way of hydrophobic interactions, against the helix of ZF2, thereby positioning the

Fig. 1. (A) Sequence of oligonucleotide containing the KBS motif (TCCT- GCNA). The box shows the core sequence equivalent to the methylated DNA site shown in B.(B) Sequence of the MeECad oligonucleotide containing two symmetrically methylated CpG sites (boxed). (C) Overlay of the Kaiso:KBS (dark colors, yellow DNA) and Kaiso:MeECad (light colors, gray DNA) X-ray

crystal structures, superimposed on the protein backbone. The three Cys2His2 zinc finger DNA-binding domains are colored red (ZF1), green (ZF2), and blue (ZF3), and the zinc atoms are shown as gray spheres. The C-terminal extension is colored purple. In the crystal structure of the KBS complex, the protein backbone is ordered between residues 482–597, whereas the MeECad complex is ordered between residues 481–600. (D) Superposition of the crystal structure (red protein and DNA) and the 20 lowest-energy structures in the NMR ensemble of the Kaiso:KBS complex. The structures are superimposed on their protein backbones. The zinc finger domains in the NMR structures are colored pink (N-terminal extension and ZF1), pale green (ZF2), pale blue (ZF3), and magenta (C-terminal extension). This figure was prepared using PYMol.

diffraction and refinement statistics are summarized in Table S1. The two crystal structures are superimposed in Fig. 1C. The solution structure of the Kaiso:KBS complex (Fig. 1D) was determined from experimentally derived distance and dihedral angle restraints (SI Methods and Table S2). The overall ar- rangement of the three zinc fingers (ZF1–3) of the Kaiso DNA- binding domain is very similar in the KBS and MeECad complexes (backbone rmsd between the crystal structures is 0.50 Å). ZF2 adopts the canonical ββα zinc finger fold, whereas ZF1 and ZF3 contain three-stranded β-sheets with topology βββα and ββαβ, respectively (Fig. S1). The three Kaiso zinc fingers wrap around the DNA with their α-helices in the major groove, con- tacting only five or six base pairs in total (Fig. 1); this interaction contrasts markedly with canonical Cys2His2 zinc finger proteins, which typically contact three base pairs per finger (29).

DNA Binding Induces Structure in the C Terminus. The ββαβ topology of ZF3 appears to be unique; residues 583–586, C-terminal to the 1 15 ZF3 helix, fold back along the DNA phosphate backbone to form Fig. 2. (A) H- N HSQC spectra of free (black) and KBS-bound (red) Kaiso. the third β-strand of ZF3 (Fig. S1). NMR experiments indicate that Selected cross peaks are labeled to indicate the large changes in chemical shift – that occur as the C-terminal extension becomes structured upon binding to the C-terminal region, residues 575 604, is intrinsically disordered DNA. (B) {1H}-15N heteronuclear NOEs for free Kaiso (black squares) and Kaiso in the free protein and undergoes a conformational change to in complex with KBS (red triangles). The small values of the heteronuclear NOE a more ordered state upon binding to DNA. The cross peaks of and the nearly random coil chemical shifts for residues 575–604 in the free 1 15 many of these residues lie in the random coil region of the H- N protein indicate that this region is disordered in the absence of DNA.

15230 | www.pnas.org/cgi/doi/10.1073/pnas.1213726109 Buck-Koehntop et al. Downloaded by guest on September 27, 2021 The C-terminal extension to ZF3, which is intrinsically disordered in the absence of DNA, is absolutely essential for high- affinity DNA binding. Truncation of the zinc finger construct at Gly-579, following the ZF3 helix, severely impairs KBS binding (10). The structures show that this impairment results from the elimination of DNA backbone contacts (from Tyr-584 and Leu- 586) and the disruption of an extensive network of minor groove interactions. In the crystal structures of both complexes, the Arg- 595 side chain is inserted deeply into the minor groove opposite the core-recognition site, where it forms hydrogen bonds with the O2 of 5mC28 and the N3 of A27 in MeECad (Fig. 4A) and with the O2 of T26 and the N3 of G27 in KBS (Fig. 4B). In the MeECad complex, the aromatic rings of Tyr-597 and Tyr-599 are inserted deeply into the narrow minor groove at the core mCpG site, where they pack tightly against the sugars of T12, C13, and G29. The OηH atom of the Tyr-597 side chain forms a hydrogen bond to the N2 amino group of G11, an interaction that is but- tressed by an additional hydrogen bond to the Arg-595 guanidinium group. The Ala-598 amide hydrogen bonds to the phosphate backbone of C13, helping anchor the neighboring Tyr residues in the minor groove. In the crystal structure of the KBS complex, the location of the two Tyr side chains is indeterminate, because no electron density is observed for residues beyond position 597, and no side-chain density is observed for Gln-596 or Tyr-597. In solution, however, intense intermolecular NOEs are observed between the ribose protons of A13/G28 and the aromatic protons of Tyr-597/Tyr-599, showing that these side chains do in fact bind in the minor groove of the KBS DNA (Fig. 4C and Fig. S3B). We conclude that the C-terminal extension assumes essentially the same conformation in the two complexes, making extensive minor groove contacts that are essential for high-affinity binding to both the mCpG and the KBS sites. Base-specific interactions are dominated by side chains in the N-terminal regions of the ZF1 and ZF2 helices, whereas ZF3 interacts primarily with the phosphate backbone and makes no (MeECad) or few (KBS) base-specific contacts. In both the crystal and NMR structures of the KBS complex (Fig. 1), devi- ations from regular B-form geometry in the core-recognition site (6TCCTGCCA13) allow the ZF3 helix to penetrate the major groove so that the side chain of Gln-563 can form direct and water-mediated hydrogen bonds to the bases of G32 and C7 (Table S3). In contrast, because of differences in DNA conformation, the ZF3 helix is unable to extend deeply enough into the major groove of the MeECad DNA to form base-specific con- ε tacts (Fig. 1A). Instead, N 2 of the Gln-563 side chain forms a hydrogen bond to the phosphate backbone of C5 (Table S3). Overall, the area of the major groove contacted by ZF3 is smaller in the MeECad complex than in the complex with KBS. Recognition of the KBS and mCpG sites by Kaiso is accom- Fig. 3. (A) DNA backbone contacts between ZF1 (magenta) and ZF2 (green) plished through base-specific hydrogen bonding and hydrophobic and the coding strand of the KBS complex. Cyan spheres indicate water mol- interactions with the C5-methyl groups of thymines (KBS) or ecules that mediate hydrogen bonding to the phosphate backbone. (B) methylated cytosines (MeECad) within the major groove (Fig. Interactions of ZF2 (green) and ZF3 (blue) with the backbone of the noncoding A strand in the MeECad complex. ZF3 is anchored to the DNA backbone by 5 ). In the MeECad complex, residues in the N-terminal regions a network of hydrogen bonds from a cluster of tyrosines (Tyr-550, Tyr-562, and of the ZF1 and ZF2 helices provide hydrophobic environments Tyr-584) and from the Arg-549 guanidinium group. The side chains of Arg-595, for the C5-methyls of 5mC10/5mC28 and 5mC8/5mC30, re- γ Tyr-597, and Tyr-599 in the C-terminal extension (pink) project deeply into spectively. The Cys-505 side chain and the C -methyl of Thr-507 the narrowed minor groove. This figure was prepared using PyMol. form a hydrophobic pocket that accommodates the C5-methyl group of 5mC10 (Fig. 5B); if the cytosine were unmethylated, the

distance between Cys-505 and the base edge would be greater, BIOCHEMISTRY C-terminal region of the polypeptide chain to enter the minor and the effectiveness of these hydrophobic interactions would be B A γ groove (Fig. 3 and Fig. S3 ). A notable difference between the diminished. The Thr-507 C -methyl also packs against the C5- X-ray and NMR structures is observed at the N terminus. There is methyl of 5mC28 (Fig. 5B). Similar interactions are observed in no electron density for residues preceding the N-terminal β-strand the KBS complex, through a change in the side-chain rotamer γ of ZF1 in either crystal structure. The conformation of this region that places the Thr-507 C -methyl in contact with the T26 C5- also is disordered in the NMR structural ensemble, but weak in- methyl and leads to the formation of a water-mediated hydrogen γ termolecular NOEs from the Arg-475 and Lys-477 side chains in- bond between Thr-507 O and the N4-amino of C11 (Fig. 5C). A dicate transient interactions with the DNA backbone across the further contribution to speciﬁcity comes from hydrophobic δ minor groove (Fig. 1D). interactions with the C 2-methyl of Leu-533, which lies close to

Buck-Koehntop et al. PNAS | September 18, 2012 | vol. 109 | no. 38 | 15231 Downloaded by guest on September 27, 2021 Fig. 4. Minor groove interactions. (A) View of minor groove protein:DNA contacts for the MeECad complex, showing hydrogen bonding (dashed red lines) between the Arg-595 and Tyr-597 side chains and the bases of A27, 5mC28 (orange), and G11. A buttressing hydrogen bond between Tyr-597 and Arg-595 is shown in green. The protein backbone is cut away for clarity. (B) View of minor groove contacts in the KBS complex showing hydrogen bonding of the Arg- 595 side chain to T26 and G27. (C) DNA contacts made by C-terminal extension [purple backbone with nitrogen (red) and oxygen (blue) molecules] in NMR structures of the Kaiso:KBS complex, showing the insertion of Arg-595, Tyr-597, and Tyr-599 side chains into the minor groove (DNA in beige with nitrogen in blue and oxygen in red). This ﬁgure was prepared using PyMol.

the C5-methyl groups of 5mC8 and T9 in the MeECad and KBS requires that a guanine occupy both positions, irrespective of the complexes, respectively. methylation status of the base-paired cytosine. Although Arg-511 There are marked similarities in the hydrogen-bonding inter- cannot distinguish 5mC from C, it clearly discriminates against actions with bases in the major groove of the core MeECad and ApT or TpA steps and thus plays a key role in recognition of the KBS sites, despite differences in sequence and cytosine methyl- central G9:mC30/mC10:G29 base pairs. In the KBS complex, ation. These interactions are mediated almost entirely by the side Arg-511 forms similar side-chain hydrogen bonds to the O6/N7 of chains of Glu-535 (ZF2) and Arg-511 (ZF1). These residues are G10 and the O6 of G28 (Fig. 5E); substitution of either guanine invariant in Kaiso sequences from different species and in the by thymine greatly impairs binding (9). The importance of these Kaiso-like zinc finger proteins ZBTB4 and ZBTB38 (30), sug- hydrogen bonds for all DNA-binding interactions is demonstrated gesting that these proteins all share a common mechanism of by mutagenesis (to alanine) of the equivalent Arg residue (Arg- DNA recognition. The Glu-535 side chain plays a critical role in 326) in the Kaiso homolog ZBTB4, which abrogates binding to the recognition of the symmetrically methylated mCpG site in both methylated and unmethylated DNA (31). Comparison of the MeECad by forming CH···O hydrogen bonds with the C5- KBS and MeECad complexes shows that as long as the Arg-511/ methyls of these bases (Fig. 5D and Fig. S4). The marked de- guanine hydrogen bonds are maintained, Kaiso can tolerate crease in binding affinity observed when all of the 5mC bases are thymine substitutions at the 5mC8 position (equivalent to T9 in replaced with nonmethylated cytosines (7, 10) confirms the im- KBS) and likely at 5mC28, although substitution of either 5mC10 portance of the methyl CH···O hydrogen bonds in specific rec- or 5mC30 with a thymine would greatly impair binding (9). ognition of mCpG sites by Kaiso. Methyl CH···O hydrogen bonds appear to be common in 5-mC recognition, because they have Recognition of Hemimethylated DNA. To assess the extent to which been observed in complexes of MeCP2 and UHRF1 with each 5mC site contributes to recognition, we examined the binding methylated or hemimethylated DNA, respectively (22, 23, 25). of Kaiso to hemimethylated and single-site symmetrically meth- Interestingly, the position of the Glu-535 side chain is virtually ylated DNA. Kaiso binds equally well to MeECad and to a probe identical in the KBS complex of Kaiso, where it forms a hydro- methylated only on the coding strand (5mC8/5mC10), whereas gen bond with the N4-amino group of C29 as well as a methyl methylation on only the noncoding strand (5mC28/5mC30) CH···O hydrogen bond with the C5-methyl of T9 (Fig. 5D and reduces the affinity slightly (Fig. S6 A and B). Additionally, sym- ε Fig. S4). The Glu-535 O 2 atom is surprisingly close (2.7 Å) to metric methylation at a single site decreases the binding affinity the O4 atom of T9, suggesting formation of a hydrogen bond only slightly, by ∼1.5-fold for the 5′-site (5mC8/5mC30) and through protonation of the carboxylic side chain. A comparison ∼twofold for the 5mC10/5mC28 site (Fig. S6C). Thus, Kaiso binds of the NMR spectrum of Kaiso in complex with the two different with significant affinity to hemimethylated DNA and to a single DNA sequences (Fig. S5) shows a large difference in the 15N mCpG site within the context of the ECad sequence. Binding to amide chemical shifts of Glu-535 and Tyr-536, likely reflecting hemimethylated DNA appears to be asymmetric, with a slight a change in the Glu-535 protonation state between the two preference for methylation on the coding strand. Similar behavior complexes. The hydrogen-bonding interactions of Arg-511 and has been observed for the Kaiso-like protein ZBTB4 (24, 31), Glu-535 are poorly defined in the NMR ensemble because of suggesting a common mechanism by which Kaiso family members a lack of intermolecular NOEs between their side chains and read and interpret the methylation signal. the DNA. Base-specific interactions involving the guanidinium of Arg-511 Conclusions also are similar in the two complexes (Fig. 5E). In the MeECad Our work has shown how the three Cys2His2 zinc fingers and complex, Arg-511 forms asymmetrical hydrogen-bonding contacts flanking regions of Kaiso are uniquely adapted for recognition of across the major groove with the guanines of the central two mC: both mCpG and specific nonmethylated DNA sequences. Al- G base pairs (O6 atoms of G9 and G29). This observation pro- though the mode of DNA binding, with three zinc fingers in the vides insights into why Kaiso prefers two consecutive mCpG base major groove, is generally similar to other Cys2His2 zinc finger pairs at the center of the recognition site or, at the very least, proteins, Kaiso differs markedly in the length of its core-

15232 | www.pnas.org/cgi/doi/10.1073/pnas.1213726109 Buck-Koehntop et al. Downloaded by guest on September 27, 2021 a single symmetrical mCpG site (24–26). Kaiso is anchored to the DNA by a plethora of phosphate backbone contacts that un- doubtedly contribute to binding affinity. Major groove contacts with the four or five base pairs in the core DNA sequences are mediated entirely by side chains within ZF1 and ZF2. The sequences of these two zinc fingers are strongly conserved between Kaiso and its ZBTB4 and ZBTB38 homologs, but the sequence of ZF3 has diverged considerably (30), suggesting that the third zinc finger of the various family members may interact differently with DNA or with other proteins. The minor groove interactions, made by residues in the C-terminal extension following ZF3, are required for high-affinity binding to both KBS and methylated DNA (10). The present structural analysis reveals the mechanism of DNA recognition by the Kaiso zinc finger domain and represents a first step toward a molecular-level description of the complex pathways involved in methylation-dependent silencing of tumor- suppressor genes and in the regulation of the Wnt signaling pathway by Kaiso (14, 32). Once bound to DNA, Kaiso represses the target gene by recruiting an N-CoR/histone deacetylase complex through its N-terminal BTB/POZ domain (33). Further studies will be required to elucidate the complex network of downstream protein–protein interactions through which Kaiso regulates gene expression in development and carcinogenesis. Methods Sample Preparation. Kaiso-ZF123(472-604) uniformly labeled with 15N, 15N/13C, or 15N/13C/2H was expressed and purified, and protein–DNA complexes were prepared as described previously (10). Methylated and unmethylated oligonucleotides were prepared synthetically (IDT Inc.) and dissolved in 10 mM Tris (pH 7.0). Equimolar amounts of complementary oligonucleotides were heated to 90 °C and annealed by slow cooling to room temperature to form duplex

DNA. All samples of the complex were prepared in argon-saturated 10 mM d11- Tris buffer (pH 7.0) containing 1 mM Tris(2-carboxyethyl)phosphine, 0.005%

(wt/vol) NaN3, and 5% (vol/vol) D2O (10). Complete formation of the 1:1 Kaiso: MeECad and Kaiso:KBS complexes used for NMR and crystallography was veriﬁed from 1D NMR spectra of the DNA imino proton resonances and 2D 15N-1H HSQC spectra of protein amide resonances.

EMSA. EMSA experiments were run on 5% nondenaturing acrylamide gels using 5′-end 32P-labeled duplex DNA sequences. Experimental details are Fig. 5. Kaiso:KBS and Kaiso:MeECad base-specific interactions. (A)Summary given in SI Methods. of major groove interactions. Recognition of MeECad is mediated by major groove interactions with only the four 5mC:G base pairs; additional minor X-Ray Structure Determination. Crystals of the Kaiso:DNA complexes were groove hydrogen bonds extend the binding site to A27. The contact surface obtained using sitting-drop vapor diffusion. Crystallographic data were for KBS is larger because of the bending of the DNA around the core recog- collected on cryo-cooled crystals at the Advanced Photon Source (APS) nition sequence. The dashed box highlights the core contact site in the two beamline 23ID-D and the Stanford Synchrotron Radiation Lightsource (SSRL) DNA sequences. Residues are colored red, green, blue, and purple to denote beamline 9-2 for the KBS and MeECad complexes, respectively. The Kaiso:KBS theirlocationinZF1,ZF2,ZF3,andtheC-terminalextension,respectively. complex crystal structure was solved by zinc single-wavelength anomalous Residues in the C-terminal extension (purple) make minor groove contacts. dispersion and refined to 2.4-Å resolution. The crystal structure of the Kaiso: Black arrows denote hydrophobic interactions, pink arrows indicated classical MeECad complex was solved by molecular replacement using the Kaiso from hydrogen bonds, and dashed blue arrows indicate water-mediated hydrogen the Kaiso:KBS crystal structure as a search model and was refined to 2.8 Å. A bonds. A complete summary of all DNA base and backbone contacts is shown more comprehensive description of the methods and refinement procedure in Fig. S2 and Table S3.(B) Cys-505 and Thr-507 form a hydrophobic pocket can be found in SI Methods. The crystallographic and refinement statistics that accommodates the methyl groups (orange spheres) of 5mC10 and 5mC28. are summarized in Table S1. The 5-methylcytosines are highlighted in orange. (C) Base-specific interactions of Thr-507 in the KBS complex. The red lines show water-mediated hydrogen NMR Spectroscopy. NMR spectra of free Kaiso and of the KBS and MeECad bonds to the N4-amino of C11. The water is shown as a cyan sphere. (D)Hy- complexes were recorded at 298K on Bruker 600, 750, 800, and 900 MHz drogen-bonding interactions involving Glu-535 in the core mCpG- (gray) and spectrometers. Assignments of backbone and side-chain resonances were KBS- (orange) binding sites. (E) Hydrogen-bonding interactions involving Arg- made using standard multidimensional NMR experiments. Structures were 511 in the core mCpG- (gray) and KBS- (orange) binding sites. The Kaiso color determined using experimentally derived distance, dihedral angle, and re- fi scheme is as in Fig. 1C.This gure was prepared using PyMol. sidual dipolar coupling restraints. Initial structures were generated using the

program CYANA (34) and were refined using restrained molecular dynamics BIOCHEMISTRY simulated annealing using the Amber 10 software package (35). The ex- recognition site. Canonical three-zinc finger proteins “read” the perimental restraints and refinement statistics for the NMR structure en- sequence in the major groove through hydrogen bonding and hy- semble are summarized in Table S2. Details of the assignment procedure and drophobic contacts with nine or 10 base pairs (29). In contrast, structure calculations are given in the SI Methods. Kaiso has evolved to recognize a highly localized site, containing only four or five base pairs, through hydrogen-bonding interactions ACKNOWLEDGMENTS. We thank Joel Gottesfeld for assistance with EMSA in both the major and minor grooves. Like Kaiso, the MBD family experiments, Gerard Kroon for assistance with NMR experiments, and Brian M. Lee, Brendan Borin, Chul Won Lee, and Christine Beuck for helpful dis- proteins MBD1, MeCP2, and MBD2 bind to a highly localized site, cussions. X-ray data for 4F6M were collected at the Advanced Photon Source making minimal major groove contacts in the immediate vicinity of General Medicine/Cancer (GM/CA) beamline 23ID-D. The GM/CA Collaborative

Buck-Koehntop et al. PNAS | September 18, 2012 | vol. 109 | no. 38 | 15233 Downloaded by guest on September 27, 2021 Access Team has been funded in whole or in part with federal funds through operated for the DOE Office of Science by Stanford University. The SSRL National Cancer Institute Grant Y1-CO-1020 and National Institute of General Structural Molecular Biology Program is supported by the DOE Office of Bi- Medical Science Grant Y1-GM-1104. Use of the Advanced Photon Source was ological and Environmental Research, by the National Institutes of Health supported by the US Department of Energy (DOE), Basic Energy Sciences, (NIH), National Center for Research Resources, Biomedical Technology Pro- Office of Science, under Contract DE-AC02-06CH11357. X-ray data for 4F6N gram Grant P41RR001209, and by the National Institute of General Medical were collected at Beamline 9-2 of the Stanford Synchrotron Radiation Light- Sciences. This work was supported by the NIH Grant GM036643 and the source (SSRL), a Directorate of the Stanford Linear Accelerator Center Skaggs Institute of Chemical Biology. B.A.B.-K. was supported by Grant PF- National Accelerator Laboratory and an Office of Science User Facility 07-124-01-GMC from the American Cancer Society.

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