Peptide recognition by heterochromatin protein 1 (HP1) chromoshadow domains revisited: Plasticity in the pseudosymmetric histone binding site of human HP1

CSDs of HP1α/β/γ bind to the H3 fragment H3(36–58) with a similar affinity

Previous studies have shown that the CSDs of human HP1 homologs recognize a histone H3 region with a PXVXL-like motif around the DNA entry/exit sites of nucleosome but that different CSDs nevertheless behave differently (20, 21, 23). The human genome encodes three HP1 proteins (Fig. 1A), and the amino acid sequence alignment of their CSDs reveals strict conservation in 50 of 65 positions (Fig. 1B). To elucidate specific determinants of their reportedly varying interactions, we performed ITC assays with a histone H3-derived synthetic peptide containing residues 36–58 (H3(36–58)) and recombinant CSDs of HP1α, HP1β, or HP1γ (hereafter referred to as CSD_α, CSD_β, and CSD_γ, respectively). All of these CSDs bound to the tested H3 peptide with similar affinities (Fig. 1C), which is not surprising given their sequence conservation.

Engagement of CTE strengthens binding

A previous study showed that the CTE plays a role in CSD selectivity and affinity (25). To explore the role of CTE in the interaction with H3 peptides, we performed a series of ITC binding assays using CSD_α/_β/_γ of different C-terminal truncations against the histone H3 peptide H3(36–58). Our binding results support the following three conclusions. 1) The CSD without the CTE is sufficient for binding to the histone H3 fragment. CSD_α (aa 107–179), CSD_β (aa 104–174), and CSD_γ (aa 110–176), which miss most or all of the CTE, bound to the H3 peptide with comparable affinities (Fig. 1, B and C). H3 binding to these CSDs is reminiscent of PXVXL motif binding by CAF1 in that CAF1 also binds to C-terminally truncated HP1α (22). 2) Compared with the C-terminally truncated CSD constructs, constructs with the complete CTE bound to the histone H3 fragment 2–10-fold more tightly (Fig. 1D), which is consistent with the published pull-down results showing the contribution of the CTE of CSD_α to histone binding (22) and also with the previously reported role of the CTE in the interaction between the CSD of DmHP1 and its partners (25). 3) An intact CSD is essential for histone H3 binding. The CSD constructs, which lack the last three residues of the CSD (truncated at Thr-173 of CSD_α or corresponding residues of CSD_β or CSD_γ), showed no interaction in our assay (Fig. 1, B and D). Taken together, these data indicate that although the CSD without CTE is sufficient for the interaction with histone H3, the CTE strengthens this interaction significantly.

Shorter H3 peptide binds to CSDs less tightly

To measure the effect of the length of histone H3 peptides on the interaction, we performed ITC assays with a shorter H3 peptide (residues 38–52, H3(38–52)) and different CSD constructs. The shorter peptide bound to CSDs less tightly (Fig. 1D), indicating that the residues flanking the PXVXL-like motif also contribute to CSD binding, consistent with the findings of Mendez et al. (25) that the flanking residues of the PXVXL motif affected the binding affinity. Thus, CSDs interact with H3 in a manner preserving some aspects of the CSD interactions with PXVXL motif-containing proteins.

Crystal structure of HP1γ CSD in complex with H3(38–52) peptide

Although the interaction between the CSD and histone H3 has been studied previously (20–23), the molecular mechanism of this interaction is still unknown. We attempted to crystallize several CSD constructs of human HP1 proteins in complex with H3 peptides encompassing either residues 36–58 or 38–52 and succeeded in determining the crystal structure of CSD_γ (residues 110–176) in complex with H3(38–52) (Fig. 2 and Table 1).

The asymmetric unit of the CSD_γ-H3 model comprises two CSD_γ molecules (CSD-1 and CSD-2) that form a dimer hinged at their C-terminal α helices (Fig. 2, A–C). Each CSD adopts the canonical conformation of a triple-stranded antiparallel β-sheet, followed by two α helices packed against one side of the β-sheet (Fig. 2A) (26). The H3 peptide, in an extended β-strand-like conformation, is sandwiched by the C termini of both CSD monomers to form another triple-stranded β-sheet, similar to other CSD-peptide complexes (Fig. 2A) (25, 27–29). Analogous to other CSD-peptide complexes, the H3 peptide binds to the CSD_γ mainly through the hydrophobic interaction (Fig. 2B) (25, 27–29). The backbones of the H3 peptide-bound and H3 peptide-free (PDB code 3KUP) CSD_γ dimers can be superimposed (30) with root mean square deviations around 0.6 and 1.0 Å for the CSD chain A alone and the A/B dimer, respectively, indicating that binding of the peptide does not induce large conformational changes in the protein's backbone or the dimer assembly.

Resemblance to the CSD-PXVXL interaction but apparent non-equivalence of binding of prolyl residues between the histone H3 and the canonical PXVXL motif

We were able to model all residues of the H3(38–52) peptide, although some residues at the N terminus of the peptide have weak density (Fig. 2D). Residues 44–46 (G-T-V) of histone H3 pair with the β strand formed by CSD-2 residues 172–174 (Leu-Thr-Trp; hereafter we refer to H3 residues with single letters and to CSD residues with three-letter abbreviations) antiparallelly, and the H3 residues 46–49 (V-A-L) pack against the corresponding β strand from CSD-1 in parallel (Fig. 2C).

To facilitate the comparison, we adopt notation previously introduced for the CSD-CAF1 complex (27) and designate H3V46 as position 0 (Fig. 2B). Thus, H3V46 is located near the pseudosymmetry axis of the CSD dimer and its side chain is accommodated by a hydrophobic pocket formed by both CSD monomers' Tyr-168 and Leu-172 residues (Fig. 2, C and E). The V46A mutant would interact less effectively with the hydrophobic pocket, consistent with published work (Fig. 2B) (21, 27), whereas the mutation of the corresponding V residue in other PXVXL motifs to E or D probably cannot be accommodated by this small pocket and destroys the interaction between CSD and its partners (11, 12, 31, 32). H3T45 and H3A47 correspond to −1 and +1 of the PXVXL motif, respectively. The side chains of these residues point away from the binding interface, and only the side chain of H3T45 hydrogen bonds with the side chains of His-175 from CSD-2 and of Thr-173 from CSD-1 (Fig. 2C). Hydrogen bonding would be disrupted by the hypothetical phosphorylation of H3T45 due to modified charge distribution, loss of a potential hydrogen bond donor, and/or added steric bulk. In the known CSD-PXVXL complexes, positions −2 and +2 can be occupied by large hydrophobic residues and significantly contribute to the CSD-PXVXL interaction (25, 27–29). In our complex structure, whereas L48 at +2 also inserts into a hydrophobic pocket formed by Leu-172 and Trp-174 of CSD-1 and Ala-129, Phe-167, and Arg-171 of CSD-2 (Fig. 2, C and E), position −2 corresponds to G44. Lacking a side chain, G44 fails to fully exploit the −2 binding pocket of the CSD dimer (Fig. 2C), suggesting that the interaction at −2 may not be as important as previously thought (14, 27, 28). The carbonyl oxygen of H3P43 (position −3) forms a hydrogen bond with the main chain of His-175 from CSD-2, and this hydrogen bond is also conserved in other reported CSD complex structures (Fig. 2C) (27–29).

The side chain of H3E50 (position +4) packs against Trp-174 of CSD-1 and hydrogen-bonds with the side chain of Thr-130 and with the main chain of Asp-131 of CSD-2, whereas the side chain of H3R42 (position −4), poorly resolved, points to the solvent and packs against Trp-174 of CSD-2, with a potential cation-π interaction (Fig. 2C). Residues at positions +5, +6, −5, and −6 (even −7 in the case of a bulged peptide conformation (27)), flanking the PXVXL motif, reportedly play critical roles in binding affinity and selectivity (25, 27). In the present CSD_γ-H3 model, H3I51 (position +5) is embraced by the side chains of Thr-130, Leu-139, and Leu-150 from CSD-2 (Fig. 2, C and E). JAK2 phosphorylation target H3Y41 (position −5) (20), only vaguely resolved by electron density, appears surrounded by residues Ile-127, Thr-130, and Leu-139 from CSD-1 (Fig. 2, C and E). Our crystal structure does not conclusively resolve the structural basis for the reported rejection of phosphotyrosine in this position (20), but added bulk or modified electric charge distribution of the added phosphoryl moiety may play a role, because the H3Y41 pocket is largely hydrophobic (Fig. 2B). Accordingly, our ITC studies demonstrate significantly weaker binding of Y41-phosphorylated H3(38–52) peptide to CSD_α or CSD_γ (Fig. 1D). H3R40 (position −6), H3H39 (position −7), H3P38 (position −8), H3R49 (position +3), and H3R52 (position +6) are poorly resolved, and the nature of their interactions with the CSD, if any, is not elucidated by our crystal structure.

Structural comparison of CSDs of HP1α/β/γ explains why they have similar binding behavior

Previous studies showed that the CSDs of human HP1 homologs recognize the PXVXL-like motif containing histone H3 region differently (20, 21, 23). However, the backbone of the CSD_γ dimer superimposes (30) well with CSD_α (PDB code 3I3C) and CSD_β (PDB code 3Q6S) dimers, with root mean square deviations around 1.7 and 0.9 Å, respectively, and residues putatively interacting with H3 are strictly conserved (Fig. 1B). These structural comparisons suggest that the CSDs of HP1α/β/γ would recognize histone H3 fragment H3(36–58) similarly, which is confirmed by our ITC assay (Fig. 1D).

Combining our structural analysis and ITC assay, it is clear why removal of Trp-174, His-175, and Ser-176 of CSD_γ (or corresponding residues of CSD_α or CSD_β) would abolish binding of H3 to HP1s. The absence of Trp-174 would disrupt the binding pocket for H3L48, whereas His-175 and Ser-176 form hydrogen bonds to the H3 peptide (Figs. 1D and 2C). Previous studies also showed that residue Trp-174 plays a critical role for the interaction between CSDs and the PXVXL motif, because the W174A mutant in human HP1α (22), the corresponding W200A mutant in Drosophila HP1a (25), and the W170A mutant in mouse HP1β (26) abrogate the binding of PXVXL peptides to HP1s. Although our crystal structure excludes the CTE, it can still be reasoned that the D/E-rich CTEs (Fig. 1B) would, if present, be in close proximity to histone H3 (Fig. 2C), which is a highly positively charged protein. The negatively charged CTEs can therefore be expected to electrostatically interact with the positively charged histone H3.

Comparison with published CSD-PXVXL complexes

Whereas our model of the CSD_γ-H3 complex conserves the general architecture of CSD-PXVXL and related complexes (Fig. 3A), further inspection reveals several differences. As shown in Fig. 3A, the C termini of the PXVXL motif-containing peptides superimpose readily, whereas the N termini diverge. Like the N terminus of CAF1, the poorly resolved N terminus of the H3 peptide appears to bend toward the surface of its CSD receptor, whereas the corresponding segments of Sgo1 and EMSY peptides more closely track the respective C termini of a CSD monomer (Fig. 3A) (27–29). Because the EMSY peptides are presented in the context of a larger protein fragment, the peptides' N termini are somewhat restrained. The N terminus of the Sgo1 peptide, on the other hand, is free and the peptide forms an intermolecular, antiparallel β pair with the C terminus of the CSD and residues of the CTE (29), possibly also explaining enhanced affinity in the presence of the CTE observed in our ITC experiments and by others (22, 25).

As shown in Fig. 3, B–D, the residues at positions 0 and +2 are highly conserved and recognized by equivalent hydrophobic pockets. Despite divergent amino acid types at positions −2, +4, and +5 (Fig. 3B), PXVXL motifs interact with CSDs in a generally conserved mode (Fig. 4, A–C). However, H3G44 (position −2 of H3 peptide) suboptimally interacts with the position −2 binding pocket of the CSD dimer (see above and Figs. 2C and 4A). Thus, CSD_γ recognizes the H3 GTVAL sequence using only two of the three hydrophobic pockets that would otherwise recognize other PXVXL motifs. Outside the central motif, recognition of H3I51 by CSD_γ resembles position +5 binding between peptides with relaxed PXVXL motifs and CSDs (Fig. 4C). Equivalents of H3E50 (position +4; see above and Fig. 2C) in the EMSY_P (ENT-proximal site) (28) and Sgo1 motifs are both arginine and similarly pack against Trp-170 and hydrogen-bond with Asp-127 in CSD_β (Figs. 3B and 4B) (29). The corresponding residue in CAF1 is aspartate and hydrogen-bonds with Thr-126 in CSD_β (Fig. 3B) (27). Thus, in addition to the hydrophobic residues (25), charged residues at the periphery of the PXVXL motif could also strengthen the interaction. The corresponding residue in the EMSY_D (ENT-distal site) peptide is A118 and lacks these interactions (28), which indicates that the recognition of residues at position +4 is not universally conserved.

In addition to the conserved interactions, some other residues of the histone H3 peptide contact with CSD specifically. As mentioned above, the N termini of H3 and CAF1 peptides bend toward the interaction surface and residue H3Y41 (position −5), similar to F217/I218 (position −7/−6) of CAF1 (27), packs against several hydrophobic residues of CSD_γ to stabilize the binding, whereas the residues at position −5 of Sgo1 and EMSY more closely track the respective C termini of a CSD monomer (Fig. 4D) (28, 29). The hydrogen bonds between H3T45 (position −1) and the CSD_γ dimer may compensate for the lack of position −2 interactions (Fig. 4E). Thus, each protein ligand has its specific interactions except for the conserved hydrophobic packing to stabilize and specify the binding.

It has been known for some time that the CSD of HP1 recognizes a common motif with canonical composition of PXVXL (11). At first, valine at the pivotal 0 position of CSD complexes was thought to be invariable, as confirmed by functional, biophysical, and structural studies (11, 12, 14, 27, 31). However, proline can be tolerated at this position (28). It was also believed that prolyl, leucyl, or methionyl was required at +2 and −2 positions for CSD binding (14, 27), but the motif has been relaxed to ΦX(V/P)X(L/M/V), where Φ includes additional hydrophobic residue types (28). The present study shows that Φ may also include glycyl and presumably additional residue types, such as alanyl. Because the CSD dimer, by virtue of its symmetry, recognizes a given peptide ligand's +2 or −2 positions with chemically equivalent binding pockets, we propose to further relax the CSD binding motif to ΦX(V/P)XΦ, where Φ includes α-amino acids without side chain nitrogen or oxygen atoms. Alignment of known relevant peptide sequences appears to restrict at least one of the Φ (±2) residues to the {I, L, F} set (Fig. 3B). This definition is consistent with previous studies of the motifs found in EMSY (EMSY_D) (28), DmHP2 (25), and Sp100A (32), for example (Fig. 3B), with additional refinement of the pattern expected pending future discovery of new CSD-binding peptides. However, not all PXVXL motifs bind to the CSD, such as the PSVSL motif in HP2 (33), the PHVAL motif in STAM2a (32), and the PAVAL motif in Sgo1 (29), which demonstrates that additional factors, such as residues flanking the pivotal pentapeptide or the conformation of the peptide ligand, may also play a role.

Possible interaction model between HP1 and histone H3 within the nucleosome

Because the CSD binding motif is located near the DNA entry/exit sites within the nucleosome (Fig. 5A), the binding site is inaccessible in the static nucleosome, which is confirmed by our gel shift assay of interaction between recombinant nucleosome and CSDs of HP1 proteins (data not shown). Whereas the nucleosome structure is dynamic, the interaction site could be exposed during some biological processes, such as DNA replication or transcription, recombination, chromatin remodeling, or histone variant exchange, during which the DNA is unwrapped off the octamer surface (34–36). The previous studies have reported that the de novo assembly of HP1 to chromatin happens in the late S-phage (37), and the remodeling complexes, such as SWI/SNF and ACF1-ISWI, promote the recruitment of HP1 to chromatin (21, 38, 39). In addition, the three HP1 proteins could interact with the tailless nucleosome and specifically bind to the folded histone H3 (40). Most importantly, the study of Cheutin et al. (41) has demonstrated that both the CD and CSD of HP1 proteins could bind to native chromatin in vivo, and these two domains contribute differently to location of HP1 in the nucleus. Consistent with these findings, our pull-down assay confirms the interaction of recombinant octamer and CSDs (Fig. 5B). Based on these studies, a possible model of interaction between HP1 and histone H3 within the nucleosome is proposed (Fig. 5C). When the DNA disassembles from the nucleosome, an open nucleosome structure will benefit the loading of HP1 proteins to nucleosome via the interaction between CSDs and the GTVAL motif, and the CD domains of the HP1 dimer may bind to the H3K9me3 modified histone in the same nucleosome or the nearby nucleosome, which will condense the chromatin structure.

In conclusion, a combination of structural analyses and quantitative binding assays reveals that human HP1α/β/γ can bind to histone H3 in the narrow H3P38–H3R52 region, although an expanded H3 peptide binds more tightly. Our crystal structure suggests that H3 recognition by CSD_γ resembles CSD-PXVXL interaction, but that the proline residues of the PXVXL and PXXVXL motifs, respectively, are functionally distinct. Consequently, and in refinement of work published by others, we propose the expanded pseudopalindromic ΦX(V/P)XΦ pattern to describe both histone and non-histone sequence motifs that interact with CSD homodimers. The monomers of the CSD homodimer cooperatively bind to peptides that, due to their unidirectionality, cannot perfectly complement the homodimer's symmetry. The enabling plastic capacity of the homodimer suggests that additional protein ligands of CSDs may remain to be discovered.

Protein expression and purification

The CSDs of HP1α (residues 109–191, 107–179, and 107–173), HP1β (residues 106–185, 104–174, and 104–169), and HP1γ (residues 109–183, 110–176, and 110–173) were subcloned into a modified pET28-MHL vector. The encoded N-terminal His-tagged fusion protein was overexpressed in Escherichia coli BL21 (DE3) codon plus RIL (Stratagene) cells at 15 °C and purified by affinity chromatography on nickel-nitrilotriacetate resin (Qiagen), followed by tobacco etch virus protease treatment to remove the tag. Protein was further purified by Superdex75 gel filtration (GE Healthcare). For crystallization experiments, purified protein was concentrated to 20 mg/ml in a buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, and 1 mm DTT. The molecular weights of purified proteins were determined by mass spectrometry.

ITC

All histone peptides were synthesized by Peptide 2.0 Inc. For ITC measurements, the concentrated proteins were diluted in 20 mm Tris, pH 7.5, and 150 mm NaCl. Lyophilized peptides were dissolved in the same buffer, and pH was adjusted by adding NaOH. Because all of the used peptides contained tyrosine, the concentrations were estimated with absorbance spectroscopy using the extinction coefficient, ϵ₂₈₀ = 1280 m⁻¹ cm⁻¹. All measurements were performed at 25 °C, using a VP-ITC microcalorimeter. Protein with a concentration of 50–100 μm was placed in the chamber, and peptide with a concentration of 0.5–1 mm was injected in 25 successive steps with a spacing of 180 s and a reference power of 13 μcal/s. Control experiments were performed under identical conditions to determine the heat signals that arise from injection of the peptides into the buffer. Data were fitted using the single-site binding model within the Origin software package (MicroCal, Inc.).

Pull-down assays

The purified His-tagged fusion octamer (30 μg) was bound to nickel-nitrilotriacetate resin (Qiagen) and mixed with the purified, tag-removed CSDs (100 μg) in a buffer containing 20 mm Tris, pH 8.0, 150 mm NaCl, 1 mm DTT for 1 h at 25 °C. After washing five times with the buffer described above, recovered proteins were loaded to SDS-polyacrylamide gels and stained by Coomassie Brilliant Blue.

Crystallization

CSD_γ (residues 110–176) was mixed with H3(38–52) at a 1:3 stoichiometric ratio and crystallized using the sitting drop vapor diffusion method at 20 °C by mixing 0.5 μl of protein solution with 0.5 μl of reservoir solution with 25% PEG 3350, 0.2 m NaCl, 0.1 m Hepes, pH 7.5, 5% ethylene glycol. Before flash-freezing in liquid nitrogen, the crystal was soaked in a cryoprotectant consisting of 85% reservoir solution and 15% ethylene glycol.

Data collection and structure determination

Diffraction data were collected under cooling to 100 K at beam line 19-ID of the Advanced Photon Source and reduced with DENZO/SCALEPACK (42) or, for later steps of refinement, with XDS (43)/POINTLESS/AIMLESS (44) software. Molecular replacement was performed with PHASER (45) and coordinates from PDB entry 3KUP. Geometric restraints for terminally protected peptide residues were prepared with GRADE (46, 47). The current model was obtained through iterative manual rebuilding with COOT (48), restrained refinement with REFMAC (49), and model validation with MOLPROBITY (50). Anisotropic displacement parameters were analyzed on the PARVATI server (51). CCP4 (52), PHENIX (53), and PDB_EXTRACT (54) programs and the IOTBX library (55) were used in preparing the model summary (Table 1) and PDB deposition.