Structural Basis for Specific Binding of Human MPP8 Chromodomain to Histone H3 Methylated at Lysine 9

Background M-phase phosphoprotein 8 (MPP8) was initially identified to be a component of the RanBPM-containing large protein complex, and has recently been shown to bind to methylated H3K9 both in vivo and in vitro. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. Methodology/Principal Findings Here, we reported the crystal structures of human MPP8 chromodomain alone and in complex with the trimethylated histone H3K9 peptide (residue 1–15). The complex structure unveils that the human MPP8 chromodomain binds methylated H3K9 through a conserved recognition mechanism, which was also observed in Drosophila HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two β strands from the two protomer subunits. Conclusions/Significance Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore.

The M-phase phosphoprotein 8 (MPP8), which was firstly identified to coimmunoprecipitate with the RanBPM-comprised large protein complex, was shown to associate with methylated H3K9 both in vivo and in vitro [33,34,35]. The binding of MPP8 to methylated H3K9 recruited the H3K9 methyltransferases GLP and ESET, as well as DNA methyltransferase 3A (DNMT3A) to the promoter of the E-cadherin gene, a key regulator of tumor cell growth and epithelial-to-mesenchymal transition (EMT) [36,37]. The recruitment of those enzymes and enzyme complexes, which regulated the H3K9 and DNA methylation at the promoter of Ecadherin gene, respectively, repressed the tumor suppressor gene expression and, in turn, played an important role in epithelial-tomesenchymal transition and metastasis [34].

Overall structure of hMPP8 chromodomain
To unveil the molecular architecture of the chromodomain of hMPP8, hMPP8 chromodomain (55-116 residues) was recombinantly expressed and crystallized. The crystals of the free-hMPP8 and hMPP8-H3K9me3 complex both diffracted to 2.05 Å resolution and the structures were solved using molecular replacement. The quality of the X-ray diffraction data and the structure refinement parameters are shown in Table 1.
In the free form, the hMPP8 chromodomain consists of a twisted anti-parallel b-sheet formed by three b-strands (named b2-b4), and a helix (named aA) located at the C-terminal end packing against one edge of the b-sheet next to b2 (Fig. 1B). In the asymmetric unit of the crystal, two hMPP8 chromodomain monomers form a dimer through the interaction between the b2 strand from each monomer. The b2 strand from one subunit runs anti-parallel to the b2' strand from the neighboring one, pulling the three-stranded anti-parallel b-sheets of two hMPP8 chromodomain proteins adjacent to constitute a six-stranded anti-parallel b-sheet (Fig. 1B). Specifically, Asp66, Met67, Thr69, Gly71 and Gly72 of b2 strand form hydrogen bond with Gly72', Gly71', Thr69', Met67' and Asp66' of b2' strand from the opposite subunit, respectively. In addition, Asp66, Met67, Lys68, Glu70, Lys109, Ile110 and Asn113 contact Thr69', Glu70', Met67', Asn113' and Ile110' via van der waals interactions (Fig. 1C). The dimer interface has a buried surface area of about 1025 Å 2 , which is strong enough to form a stable dimer. As reported, the dHP1 chromodomain existed as monomer while dPlycomb chromodomain formed dimer both in solution and in crystal lattice [19,20,23,30]. Sequence alignment result indicates that hMPP8 chromodomain is more similar to HP1 choromodomain and lacks the residues in dPlycomb chromodomain for dimerization (Fig. 1A). It was quite unexpectedly to find that hMPP8 chromodomain forms homodimer in our crystal structures.
To determine the oligomerization state of hMPP8 chromodomain in solution, size exclusion experiment was performed. As shown in Fig. 1D, hMPP8 chromodomain eluted as a single peak with apparent molecular weight of 13.6 kD. The molecular weight of hMPP8 chromodomain monomer is about 8.0 kD. The size of hMPP8 chromodomain in solution is corresponding to dimer, consistent with the observation in crystal structure. Therefore, the chromodomain exists as a homodimer in solution (Fig. 1D) and the homodimer structure in crystal is not due to crystal packing.
Structural basis for the specific binding of the hMPP8 chromodomain to histone H3 methylated at lysine 9 Since hMPP8 chromodomain was reported to bind methylated H3K9 [34,35,38], we used synthetic di-and tri-methylated H3K9 (residues 1 to 15) peptides to measure their binding affinities to the hMPP8 chromodomain by surface plasmon resonance (SPR) method. The hMPP8 chromodomain showed strong binding to both di-and tri-methylated H3K9 with the dissociation constants of 0.43 mM and 0.31 mM, respectively ( Fig. 2A, 2B). In histone H3, the amino acid sequence around lysine 27 site (KAARK 27 S) is similar to that of the lysine 9 site (QTARK 9 S). A synthetic H3K27me3 (residues 19 to 33) peptide was also used to determine the binding affinity to hMPP8 chromodomain. However, the hMPP8 chromodomain does not exhibit detectable binding to the both H3K27me3 peptide (Fig. S1F). In addition, the binding of hMPP8 chromodomain to H3K4me3 peptide was unable to be detected (Fig. S1F), which was consistent with previous reports [31,35]. To explore how the hMPP8 chromodomain selectively binds the methyl-K9-containing histone H3 tail, we determined the crystal structure of the hMPP8 chromodomain in complex with the H3K9me3 peptide. The overall structure of the hMPP8 chromodomain in complex with the H3K9me3 peptide is shown in Fig. 2C. Two histone H3K9me3 peptides bind to the opposite faces of the hMPP8 chromodomain homodimer, respectively (Fig. 2C). Structural comparison of the hMPP8 chromodomain-H3K9me3 peptide complex and the free hMPP8 chromodomain identified a newly formed b strand (named b1) by the N-terminal residues, which exited as a loop in the free hMPP8 chromodomain structure (Fig. 2D). This b strand is induced by the contact with the H3 tail peptide, which was observed in the structures of Drosophila HP1 and Polycomb chromodomain in complex with methyllysine histone peptides before [23,24,27]. From the complex structure we can see that the H3K9me3 peptide binds to hMPP8 chromodomain in a cleft between the N-terminal newly formed b1 strand and the loop connecting b4 and aA. Similar to the structure of the Drosophila HP1 and Polycomb chromodomain in complex with methyllysine histone peptides, the interactions between hMPP8 chromodomain and H3K9me3 largely involve the main chains of both the protein and the peptide, including the residues Gln5, Thr6, Ala7, and Arg8 of the H3 tail and the residues Val58, Phe59, Glu60, and Val61 located at the b1 strand in hMPP8 chromodomain. In addition, the residues of Gln5 and Arg8 form van der waals contacts with the residues of 98-100 located in the loop connecting b4 and aA, whereas Gln5 and Ser10 form hydrogen-bonds with residues of Glu101, Val102 and Glu91, respectively (Fig. 2E). As demonstrated in most complex structures of methyllysine peptides and their recognition modules, the trimethylated K9 lies in a hydrophobic pocket formed by three aromatic residues, Phe59, Trp80, and Tyr83 (Fig. 2F). And the trimethyl-K9 is anchored by cation-p and van der Waals interactions within this aromatic cage. It is noteworthy that the sequence motif of H3K27 is similar to H3K9 tail (KAARK 27 S versus QTARK 9 S). However, methylated histone H3K27 cannot interact with hMPP8 chromodomain. To explain why hMPP8 specifically recognizes methylated H3K9, we built a mutant model that hMPP8 binds to methylated H3K27. We mutated residues Gln5 and Thr6 of the QTARK 9 S motif to KA to generate a motif of KAARK 9 S, which shared the same amino sequence around the K27 site in histone H3. In this mutant model, we found that the side chain of Lys5 prevented the insertion of histone peptide into the binding groove of hMpp8 chromodomain (Fig. S1A). To further validate the hypothesis, hMpp8 chromodomain mutants were designed to rescue the binding ability to H3K27me3. Based on our structures, residues Lys100, Glu101 and Val102 were mutated to proline, respectively, to generate enough space where the side chain of Lys5 can insert. As expected, when Lys100 or Glu101 were mutated to proline respectively, the mutant proteins were found to be able to bind H3K27me3 peptide weakly (Fig. S1B).
Furthermore, a structure model of peptide KAARK(me3)S (referred to as H3K27me3 peptide), which shared the same amino sequence around the K27 site in histone H3 was generated and docked into hMPP8 chromodomain structure using the program HADDOCK [39] (Fig. S1C). In this model, the H3K27me3 peptide can still form a b sheet and interact with hMPP8 via hydrogen-bond and van der Waals interactions. However, the side chain of trimethyllysin was pushed about 24u away from the original binding site in the hydrophobic pocket, which is essential for the interaction between methylated peptide and its association domain (Fig. S1D). Thus we believe that altering the motif of QT to KA abolishes the binding ability of H3K27me3 peptide to hMPP8 chromodomain.

Structural comparison of hMPP8 with HP1 and Polycomb chromodomains
Consistent with the high sequence homology of the hMPP8 chromodomain to the Drosophila HP1 and Polycomb chromodomains (Fig. 1A), the overall structure of hMPP8, Polycomb and HP1 chromodomains are very similar [23,24,27]. Unsurprisingly, the binding mode of hMPP8 to the methylated-H3K9 peptide is also similar to that observed in the structures of HP1 and Polycomb chromodomain in complex with the methylated histone peptides. The structure of hMPP8 chromodomain is well conserved with an RMSD of 0.8 Å and 1.1 Å for all aligned C a atoms with those of the HP1 and Polycomb chromodomain, respectively. In addition, the histone peptide conformation in the hMPP8 chromodomain complex structure is also very similar to its counterparts in the complex structures of the HP1 and Polycomb chromodomains, with an RMSD of 0.4 Å and 0.5 Å , respectively (Fig. 3A). Though the architectural features of the hMPP8, HP1 and Polycomb chromodomains are highly similar, there are still many noticeable differences among them.
The most striking difference is that the hMPP8 chromodomain was found to form homodimer in both solution and crystal lattice, which was not observed in Drosophila HP1. Although selfassociation of the chromodomain of Drosophila Polycomb has been pointed out explicitly before [27], the interaction mode of the two subunits in hMPP8 chromodomain homodimer is different from that of Polycomb. In Polycomb chromodomain homodimer, the key residues that involved in dimerization are located in the loop at C-terminus connecting the last b strand and the last a helix (Fig. 1A, 3B). However, dimerizaiton of hMPP8 chromodomain is formed via extensive intermolecular interactions between the two b2 stands from two individual subunits, including van der waals contacts and hydrogen-bonds. (Fig. 1C).
A total of 12 out of 15 residues in H3K9me3 were observed to be ordered in hMPP8 chromodomain complex corresponding to the sequence stretch from Lys 4 to Ala 15, whereas there were only 6 and 9 residues observed in the structures of Drosophila HP1 and Polycomb, respectively [23,24,27]. hMPP8 chromodomain possesses a more extended peptide binding groove than that of HP1, comparing the 1250 Å 2 of the interaction area of hMPP8 to 1063 Å 2 of HP1. Nevertheless, it is not convincing to deduce that the longer H3 tail observed in the structure of hMPP8 chromodomain complex is just because of the extended proteinpeptide interaction, since Polycomb chromodomain has the most extended peptide binding groove among the three, 1482 Å 2 (Fig. 3D). In the crystal lattice, another pattern of the homodimer of hMPP8 chromodomain was found. The two chromodomain juxtaposed the two H3-binding clefts in an antiparallel fashion and resulted in not only histone-histone interactions involving Ser10, Gly12 and Cly13 of H3, but also the interactions between histone peptide and the neighboring chromodomain involving residues 11-15 of H3 with residues leu75 and 88-90 of the adjacent chromodomain. Those additional interactions can further stabilize H3 peptide, especially residues 11-15 (Fig. 3C).
To verify whether the H3K9me3 peptide could bring the two chromodomain homodimers together in solution, size-exclusion chromatography were performed to determine the oligomerization state of hMPP8 chromodomain either in the presence or in the absence of H3K9me3 peptide. The elution volumes of hMPP8 chromodomain in free form and in complex with H3K9me3 peptide were approximately 12.62 ml and 12.60 ml, respectively, which were both corresponding to the homodimer of hMPP8 chromodomain (Fig. S1E). The results indicated that the pattern that two chromodomain juxtaposed the two H3-binding clefts in neighboring hMPP8 chromodomain dimers is only a crystalpacking artifact.

Discussion
Recently, more and more evidences have suggested that many histone-mark ''readers'' and ''writers'' can also bind non-histone sequences [40,41,42]. Chromodomain is conserved among both plants and animals, which functions individually or in tandem to recognize specific methylated histone tails [43,44]. CBX3 chromodomain have been reported to bind H3K9me3 peptide [45]. However, a recent structure of CBX3 chromodomain in complex with G9a peptide (PDB: 3DM1) demonstrates that CBX3 is also a reader of methylated G9a. Here, we resolve the structure of hMPP8 chromodomain in complex with H3K9me3 peptide and shed lights on the molecular mechanism of selective binding of hMPP8 to methylated histone H3K9. Based on our structure, we tried to mutate some residues of H3K9me3 peptide and generate structure models by docking the mutant peptides into the hMpp8 chromodomain using the program HADDOCK [39], we finally hypothesized a consensus sequence of (Q/N)(T/V/L/I/S)A(R/ K/H)Kme(S/T) (''/'' separates tolerated amino acids at each site). Such consensus sequence may be helpful to predict the candidate Mpp8-interacting proteins which could potentially be methylated.
In addition, our crystal structures reveal that hMpp8 forms homodimer via b-sheet interactions between the neighboring subunits, which are never observed in the structure of either HP1 or Polycomb chromodomain before. The distanance between the two aromatic cages binding methylated H3K9 in hMpp8 chromodomain homodimer is measured to be 40 Å , so it would be reasonable to speculate that hMPP8 chromodomain dimer may bind two methylated H3K9 from the same nucleosome or spatially adjacent nucleosomes. Here we build the models that the simultaneous binding of two histone tails to hMpp8 homodimer either from the same nucleosome or from two separated nucleosomes ( Fig. S2A and S2B). We believe that the interactions of hMPP8 homodimer with two histone H3 tails methylated at K9 are able to recruit the H3K9 methyltransferases GLP and ESET, as well as DNA methyltransferase 3A more efficiently, hereby contribute to gene repression.

Note
During preparation of this manuscript, another group reported the MPP8-K9me3 complex [46]. Cheng and colleagues also observed that hMPP8 chromodomain formed homodimer both in solution and in crystal structures. The recognition mode between hMPP8 chromodomain and methylated histone H3K9 peptide in their complex structure is almost the same as that observed by us. We both found that hMPP8 cannot recognize methylated H3K27 and the binding affinity of hMPP8 chromodomain to H3K9me3 is similar.

Protein expression and purification
The chromodomain of human MPP8 (residue 56-116) was inserted into a pET28a-MHL vector via ligase-independent cloning. The recombinant protein was expressed in BL21 (DE3) Codon plus RIL (Stratagene). Cells were grown at 37uC to OD600 of approximately 6 and protein expression was induced by 0.1 mM IPTG for another 16 hours 15uC. Cells were collected by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.4% NP40, 0.5 mM TCEP, 5 mM immedazole, 20 ul Benzonase, and protease inhibitors). The resuspended cells were lysed by sonication and centrifugated at 16000 rpm for 60 minutes at 4uC. After centrifugation, the supernatant was passed through a Ni-NTA nickel-chelating column (Qiagen) equilibrated with lysis buffer and the column was extensively washed with washing buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 25 mM immidazole, and 0.5 mM TCEP). Target protein was eluted with buffer (500 mM NaCl, 50 mM Tris, pH 8.0, 250 mM imidazole) for 3 column volumes. His-tag was removed by TEV protease. After digestion, protein sample was further purified by a HiLoad 16/60 Superdex 200 size exclusion column (GE healthcare).
Protein Crystallization, X-ray diffraction data collection and structure determination Before crystallization, the protein was concentrated to 26 mg/ ml as stock in 280uC. Crystals of hMPP8 chromodomain were obtained by the hanging drop vapour diffusion method at 18uC in a buffer containing 25%PEG400, 0.2 M MgCl2, 0.1 M Hepes 7.5. For crystallization of complex, H3K9me3 peptide was mixed with hMPP8 chromodomain in an 8:1 molecular ratio, then the mixture was crystallized using the hanging drop vapour diffusion method at 18uC. hMPP8-H3K9me3 complex was crystallized in a buffer containing 35% PEG2000-MME. Before flash-freezing crystals in liquid nitrogen, crystals were soaked in a cryoprotectant consisting of 100% reservoir solution and 15% glycerol.
Diffraction data were collected at beamline 19ID of the Advanced Photon Source (Argonne, Illinois). Data were reduced using the HKL suite [47]. Structures were solved by molecular replacement using the structure of human chromobox homolog 3 chromodomain (PDB ID: 3DM1) as template and refined with REFMAC [48]. The peptide ligands were automatically traced with BUCCANEER [49]. Interactive model rebuilding and validation were performed with COOT [50] and the MOL-PROBITY server [51], respectively. The quality of the structure models was analyzed with the PROCHECK program [52]. The coordinates and structure factors have been deposited to the RCSB Protein Data Bank with accession numbers of 3LWE and 3R93. Details can be found in table 1.

Surface Plasmon resonance (SPR) assay
The binding affinity of hMPP8 chromodomain and histone peptides were determined at 14uC using BIAcore3000 instruments. The biotinylated peptides were immobilized on a streptavidin-coated biosensor chip (SA-Chip). All experiments were carried out in the continuous-follow buffer (150 mM NaCl, 20 mM Tris, pH 8.0, 1 mM DTT). The injected protein sample was flowed for 3 min over the peptide coated SA-Chips at a follow rate of 30 ml/min and the change of response unit (RU) was recorded. Protein dissociation was monitored for 3 min by following the continuous-follow buffer at a follow rate of 30 ml/ min over the SA-Chips. The KD was determined by global nonlinear regression fitting of the association and dissociation curves according to the Langmuir binding isotherm model. Figure S1 hMPP8 specifically recognizes methylated H3K9 rather than H3K27. (A) A mutant structure model for hMPP8 binds to KAARK(me3)S histone motif. Gln5 and Thr6 of the QTARK 9 S motif were mutated to KA in this model.