A minimum HIRA fragment that binds to ASF1a
Wild-type human HIRA is a polypeptide of 1017 amino acids. Previously, we showed that residues 421–729 are sufficient for binding to the evolutionarily conserved N-terminal core domain of ASF1a, residues 1–155 (Supplementary Fig. 1a
), and for repression of histone gene expression and formation of SAHF in human cells 18,33
. This ASF1a-interacting domain of HIRA contains a stretch of about 37 amino acids that is evolutionarily conserved in HIRA and its orthologs and named the B-domain (defined as residues 439–47532
)(). The B-domain is necessary for binding to ASF1a, repression of histone gene expression and formation of SAHF18,33
. To date, the B-domain has not been described outside of the HIRA family, although the p60 subunit of CAF-1 shares some homology with this domain ().
Sequence alignment of the B-domains of HIRA and CAF-1 p60 homologues
To determine which region of the HIRA B-domain makes critical contacts with ASF1a, we made a series of scanning mutations, deleting 7–10 amino acids of the B-domain at a time. The HA-tagged HIRA proteins were tested for binding to ASF1a in vitro. The two innermost deletions (of residues 449–458 and 459–468), encompassing the most highly conserved residues of the B-domain (), had the most debilitating effect on binding to ASF1a, whereas the two outermost deletions had a more modest effect ().
ASF1a binding to HIRA deletion constructs
Having identified the regions of the B-domain that are necessary for binding to ASF1a, we next set out to define the minimum region of HIRA that is sufficient for binding. A series of HA-tagged deletion mutants derived from HIRA(421–729) was tested for binding to ASF1a. As shown in Supplementary Fig. 2
, C-terminal deletions as far as residue 509 all retained binding to ASF1a, although HA-HIRA(421–509) bound with reduced efficiency compared to HA-HIRA(421–729). An additional series of C-terminal deletions was made, this time fused at their N-terminus to GFP. As shown in , binding was retained in GFP-HIRA(421–479) but undetectable in GFP-HIRA(421–469). We conclude that the minimum region of HIRA that retains binding to ASF1a lies within amino acids 421–479, underscoring the role of the B-domain in ASF1a binding32
Next, we asked whether a short peptide spanning the most conserved amino acids of the B-domain was sufficient to interact with ASF1a, by testing its ability to compete with the binding of HA-HIRA(421–729) to ASF1a. Specifically, we tested residues 453–467 of wild-type HIRA, RTADGRRRITPLCIA, and a sequence-scrambled version, GRAARITPRDTLRCI, as a control. The wild-type peptide, but not the scrambled peptide, competed-out the interaction between ASF1a and HA-HIRA(421–729) (). Together, these results show that the B-domain is both necessary and sufficient for interaction with ASF1a.
Structure of the ASF1a/HIRA complex
Having defined the minimal region of HIRA require for Asf1a interaction, we set out to determine the X-ray crystal structure of an ASF1a/HIRA complex. Towards this goal, we coexpressed the HIRA B-domain (427–472) and the ASF1a N-terminal core domain (1–154) in E. coli
and purified the tightly associated heterodimeric complex to homogeneity. Crystals were obtained in the space group P65
22, containing two ASF1a/HIRA complexes per asymmetric unit. The structure of the complex was determined by molecular replacement using the crystal structure of the N-terminal core domain of yeast Asf1p34
as a search model. This produced a clear solution for the two ASF1a molecules in the asymmetric unit and excellent density for the two ASF1a-bound HIRA fragments (). One HIRA fragment could be confidently built into the electron density map from residues 446 to 466 and the other from residues 449 to 464 (). We presume that the remainder of the HIRA fragments is disordered. The final model was refined to 2.7 Å resolution with excellent refinement statistics ().
Overall structure of the ASF1a/HIRA complex and comparison with nascent yeast Asf1 and human ASF1a
Data Collection and Refinement Statistics
The two ASF1a N-terminal core domains in the asymmetric unit (Cα RMSD 0.45Å) adopt the same elongated immunoglobulin-like β sandwich fold () previously described for the nascent structures of human ASF1a31
and yeast Asf1p34
, with Cα RMSD of 1.1 Å relative to either structure (). However, variation between the free and HIRA-bound ASF1 polypeptides is observed at two less conserved loops (), and interestingly, also at the highly conserved β5–β6 loop at the HIRA interface (). In particular, it appears that the β5–β6 turn of free ASF1a, especially around Gly63 and Pro64, is in a conformation that interferes with HIRA binding, but adjusts to accommodate the HIRA B-domain upon complex formation.
Each of the two bound HIRA polypeptides in the asymmetric unit forms a β-hairpin containing two strands of 4 and 6 residues with a 2-residue tight turn and loop extensions N- and C-terminal to the β-strands (). In particular, residues Glu451–Thr454 and Gly457–Ile461 form a two-stranded anti-parallel β-sheet that is stabilized by 5 intra-molecular main-chain and 4 side-chain hydrogen bonds, including a bidentate salt-bridge between residues Glu451 and Arg459 (). Gly457 at the β-hairpin turn also adapts unique dihedral angles to facilitate two sets of hydrogen bonds between the backbone of Gly457 and residues Arg453 and Thr454 that stabilize the β-hairpin structure. The high degree of conservation of residues that mediate the intra-molecular interactions within the β-hairpin of the HIRA B domain, and in particular the strict conservation of Gly457 (), suggests that this structure may be preformed prior to ASF1a binding.
The B-domain of HIRA binds along the edge and perpendicular to the strands of the β-sandwich of the ASF1a N-terminal core domain and makes extensive interactions with ASF1a, burying 652Å2
of HIRA surface (). Both ASF1a/HIRA complexes in the asymmetric unit show similar contacts despite the different HIRA conformations of residues around the β-hairpin turn (). The ASF1a-binding site for HIRA is a shallow hydrophobic cleft that is primarily lined by residues Val60, Val62, Val65, Pro66, Phe72, and Phe74 from the β5–β6 region and residue Phe28 and Leu38 from the β3 and β4 strands, all involved in van der Waals interactions with residues Ile461, Pro463 and Leu464 of the HIRA B-domain (). There is also a cluster of salt bridges formed between the acidic residues Glu39, Asp58 and Asp37 of ASF1a and the basic residues Arg458, Arg459 and Arg460 of HIRA, respectively (). Finally, residues Leu61–Gly63 of ASF1a (β5) form β-sheet interactions with residues Arg459–Ile461 (β2) of HIRA. The loops before and after the β-hairpin are both secured onto ASF1a by hydrogen bonds and hydrophobic interactions (). Taken together, it appears that HIRA residues Arg458–Ile466, within the minimum ASF1a-binding peptide of HIRA (residues Arg453–Ala467) identified above, play the most critical role in ASF1a interaction. Most of the interacting ASF1a and HIRA residues involved in this interaction are either strictly or highly conserved within both proteins ( and Supplementary Fig. 1a
), highlighting the importance of these interactions.
Biochemical characterization of the ASF1a/HIRA interface
Equipped with detailed structural insight into the ASF1a/HIRA interface, we then used targeted mutagenesis and in vitro binding assays to verify the functional significance of individual interactions between ASF1a and HIRA. We first examined the importance of the salt bridge interactions. As can be seen in , alanine substitution of either the second or third arginine in Arg458–Arg460 of HIRA that interacts with D58 and D37 of ASF1a, completely abolishes binding.
Functional characterization of the ASF1a/HIRA interaction
In addition, alanine substitution of the first arginine, Arg458, partially blocks binding. Correspondingly, we probed the contribution of the respective interacting residues in ASF1a. Previously, we showed that double substitution of the two acidic residues Glu36 and Asp37 with alanine residues abolishes binding to HIRA(421–729)18
. Consistent with the structural finding that only Asp37 is involved in salt-bridge formation, the single substitution Asp37A abolishes binding to HIRA(421–729), while E36A has little effect (). Taken together, these results reveal that the charge interactions between highly conserved residues HIRA-Arg460 and ASF1a-Asp37, and HIRA-Arg459 and ASF1a-Asp58, are crucial for complex formation ( and Supplementary Fig. 1a
The role of the hydrophobic interactions between ASF1a and HIRA was also evaluated. shows that mutation of HIRA residue Ile461, one of two hydrophobic residues within the hydrophobic cleft of ASF1a, to aspartic acid abolishes the interaction between HIRA(421–729) and ASF1a. In addition, mutation of either Leu464D or Ile466D in HIRA(421–729) partially blocks ASF1a binding, and mutation of both together completely abolishes binding. Together, these findings agree with our structural findings that the hydrophobic cleft of ASF1a compliments Ile461 and Pro463 of HIRA, consistent with the strict evolutionary conservation of these two residues (). A L464D/I466D double mutant also disrupts ASF1a binding, suggesting that, although less conserved, this part of HIRA still plays a role ().
In an earlier study, we showed that mutation of the strictly conserved triplet Val-Gly-Pro to Aal-Ala-Ala (residues 62–64), located in the β5–β6 loop region of ASF1a, completely abolishes HIRA binding 34
. The structural comparison of the complex with free ASF1 proteins as described above suggested that the unique conformation and relative flexibility of this loop region is critical for proper ASF1a/HIRA interactions. This mutation, largely by eliminating the unique conformations of Gly63 and cis-Proline 64, not only disrupts direct hydrogen bonding with HIRA, but also causes potential steric occlusion of other contacts, such as the interaction between Asp37 of ASF1a and Arg460 of HIRA ( and
). Taking all our results together, we conclude that a combination of β-sheet, electrostatic and hydrophobic interactions stabilize the interaction between the ASF1a N-terminal core and the HIRA B domain.
Specificity of HIRA for ASF1a over ASF1b
In light of the earlier observations by us and Nakatani and coworkers that HIRA preferentially associates with ASF1a over ASF1b, in vitro
and in vivo18,20
, we were surprised that each of the ASF1a residues involved in HIRA B-domain binding are strictly conserved in ASF1b (Supplementary Fig. 1a
). This suggests that interactions between the ASF1a N-terminal core domain and the HIRA B-domain are not responsible for HIRA's discrimination between ASF1a and ASF1b. To test this, we measured binding of the recombinant N-terminal core domains of ASF1a and ASF1b to the minimal HIRA B-domain peptide (residues 453–467), using isothermal titration calorimetry (ITC). We also tested the full-length ASF1a protein, to evaluate the contribution of the C-terminal region of ASF1a to HIRA B-domain binding. We were unable to prepare the full-length recombinant ASF1b protein in soluble form for ITC. The ITC studies show that the HIRA peptide binds with comparable affinity to each of the ASF1 proteins, with dissociation constants in the range of 1.3–2.1 µM (). These results confirm that HIRA does not distinguish between ASF1a and ASF1b through the interaction between the HIRA B-domain and the ASF1 N-terminal core domain. It further reveals that the C-terminal region of ASF1a does not influence binding of the HIRA B-domain.
The ITC and structural studies together suggest that differential binding of ASF1a and ASF1b to HIRA might be mediated by regions outside of the HIRA B-domain and/or regions outside of the ASF1a core domain region that makes HIRA B-domain interactions. Previously, we showed that simultaneous substitution into ASF1b of two regions of ASF1a that are relatively poorly conserved in ASF1b, residues 31–37 and the C-terminal 50 residues, potentiated binding of ASF1b to HIRA18
. However subsequent studies revealed that substitution of residues 31–37 alone does not potentiate binding (data not shown). To further investigate the role of the non-conserved C-terminal tails of ASF1a and ASF1b (Supplementary Fig. 1b
) in HIRA binding, we tested binding of HA-tagged HIRA(421–729) to ASF1 chimeras in which the C-terminal tail regions of ASF1a and ASF1b were swapped (). As can be seen in (left panel)
, swapping the C-terminal tails between ASF1a and ASF1b decreases binding of ASF1a but increases binding of ASF1b. Based on these results, we conclude that, compared to the ASF1b C-terminal tail, the ASF1a C-terminus potentiates binding to HIRA.
Interestingly, in these HIRA binding studies, we observed that the ASF1a N-terminal core domain lacking the C-terminus (ASF1aN), the same region of ASF1a cocrystallized with HIRA, bound better to HIRA(421–729) than the corresponding fragment of ASF1b (ASF1bN) (, left panel
). These results, coupled with our ITC studies showing that the N-terminal core domains of ASF1a and ASF1b bound indistinguishably to the HIRA B-domain alone, suggest that another region of HIRA might bind to the N-terminal core domain of ASF1a. Of interest, the N-terminal 30 residues of ASF1a and ASF1b are less conserved than the rest of the core domain (Supplementary Fig. 1a
) and lie in the vicinity of the HIRA B-domain binding surface (see ). To test whether this part of ASF1a mediates additional contacts with HIRA outside of the B-domain and thus contributes to HIRA's specificity for ASF1a, we prepared chimeric proteins in which the N-terminal 30 residues were swapped between the ASF1a and ASF1b core domains and tested them for binding to HIRA(421–729). These experiments showed that ASF1bN carrying the N-terminal 30 residues of ASF1a binds HIRA(421–729) much better than native ASF1bN (, right panel
). Conversely, ASF1aN carrying the N-terminal 30 residues of ASF1b binds much worse to HIRA(421–729) than native ASF1aN. Based on these results, we conclude that the N-terminal 30 residues of ASF1a and ASF1b also contribute to the binding specificity for HIRA. Also, as shown in (right panel)
, ASF1aN and ASF1bN(1–30 ASF1a) have similarly high binding affinity for HIRA(421–729), while ASF1bN and ASF1aN(1–30 ASF1b) have similarly low binding affinities, suggesting that residues 31–155 of ASF1a and ASF1b do not make direct contributions to relative HIRA binding specificity.
Model for binding of other HIRA regions and the histone H3.3 C-terminal helix to the HIRA-B-domain/ASF1a complex
In sum, our binding studies show that HIRA’s discrimination between ASF1a and ASF1b depends on part(s) of the HIRA protein outside of the B-domain and both the N-terminal 30 and C-terminal 50 residues of the ASF1 proteins.
B-domain-like motifs in CAF-1 p60
Although ASF1a interacts with both HIRA and CAF-1 p60, it does not simultaneously copurify with both proteins20
. Therefore, we hypothesized that CAF-1 p60 might bind ASF1a in an analogous fashion to HIRA, using a B-domain-like motif. To test this, we searched CAF-1 p60 homologues for the consensus HIRA B-domain motif, GRRRIXPLXI, which provides the critical binding determinants for ASF1a. This search identified either one or two B-domain-like motifs in the C-terminal tail of each of CAF-1 p60 homologue in the sequence database (). Interestingly, a previous report showed that the C-terminal region of the Drosophila
CAF-1 p60 homolog (p105), which also harbors this motif, mediates ASF1 binding25
Despite their overall homology in the B-domain and B-domain-like regions, a very noticeable difference between HIRA and the CAF-1 p60 homologues is the lack of conservation of the first and, to a lesser extent, the second of the three conserved arginine residues in HIRA (residues 458–460) (), that make important salt-bridge interactions with ASF1a in the complex (). To assess the consequence of these changes, a series of mutations was generated in the 458–Arg-Arg-Arg–460 triplet of the HIRA B-domain and tested for ASF1aN binding. As shown in , single or double arginine to lysine mutations within residues 458–460 do not result in a marked perturbation of ASF1a binding, while a triple arginine to lysine change results in a more dramatic decrease. As shown in and , when Arg458 is mutated to alanine, to mimic the less polar residue found in the B-domain-like motifs in CAF-1 p60, the HIRA B-domain still maintains a relatively high level of ASF1aN binding. However, when the R458A mutation is combined with either the R459K or R460K mutations, ASF1a binding is substantially impaired, albeit not abolished (). A combination of the R458A mutation with the R459K, R460K double mutation completely abolishes ASF1aN binding (), indicating that maintaining at least one arginine (instead of lysine) at either position Arg459 or Arg460 is critical for ASF1aN binding. These results suggest that the B-domain like motifs of CAF-1 p60 and its homologues could mediate binding of the corresponding proteins to ASF1aN.
Functional characterization of ASF1a interaction with the HIRA B-domain-like motifs of CAF-1-p60
To directly test whether the two B-domain-like motifs in human CAF-1 p60 (within residues 480–490 and 496–506) mediate ASF1a binding, various CAF-1 p60 C-terminal fragments were tested for binding to the ASF1a N-terminal core domain. As shown in , CAF-1 p60 fragments containing both B-domain-like motifs (residues 376–509, 376–559, 469–509 and 469–559) interact with ASF1a, while a polypeptide with both motifs deleted (residues 376–481) shows very weak binding. A fragment harboring only the first B-domain-like motif (residues 376–496) binds with slightly reduced affinity, relative to the fragments harboring both motifs. ASF1a pull-down experiments with the yeast CAF-1 p60 homologue, Cac2p, showed that this protein also interacts with ASF1a, via a conserved B-domain-like motif within residues 457–468 (). Taken together, these studies show that human and yeast CAF-1 p60 bind to the conserved ASF1a core domain through their evolutionarily conserved C-terminal B-domain-like motifs.
To verify that ASF1a interacts with both HIRA and CAF-1 p60 through the B-domain motifs and to establish the relative importance of the first and second B-domain-like motifs in CAF-1 p60, we carried out ASF1a pull-down studies with the shortest CAF-1 p60 fragment (469–509) that contains both motifs, harboring mutations of the arginine residues in both motifs, Arg483 and Arg499, that are equivalent to the critical Arg460 in HIRA. As shown in , the R483A/R499A double mutation in CAF-1 p60 also abolishes the ASF1a interaction, while a comparable double mutation to lysine markedly compromises the interaction. Pull-down experiments with single R483A and R499A mutations of CAF-1 p60 revealed similar residual ASF1a binding capacities (), indicating that either B-domain-like motif of CAF-1 p60 can bind to ASF1a in vitro in the context of this minimal CAF-1 p60 fragment.
To test if other regions of CAF-1 p60 participate in ASF1a binding and to investigate whether both B-domain-like motifs of CAF-1 p60 contribute to ASF1a binding in the intact full-length CAF-1 p60 protein and in vivo, we carried out co-immunoprecipitation assays employing epitope-tagged CAF-1 p60 proteins ectopically expressed in U2OS cells. Specifically, we employed native full length CAF-1 p60, full length proteins containing RR to AA mutations in either the first or second B-domain-like motifs and a fragment harboring the N-terminal WD40 repeats, but not the two C-terminal B-domain-like motifs (residues 1–481). As shown in (left panel), while wild-type CAF-1 p60 exhibits strong binding activity, CAF-1 p60(1–481) is completely inactive in ASF1a binding, indicating that the N-terminal WD40 repeats are not sufficient for ASF1a binding and that the C-terminal region, harboring the two B-domain-like motifs is critical. As shown in (right panel), intact CAF-1 p60 harboring the RR to AA mutations in the second B-domain-like motif binds to ASF1a with wild-type efficiency, while mutation of the first B-domain-like motif completely abolishes binding. Taking all our results together, we conclude that in the context of the full-length CAF-1 p60 protein in vivo the first B-domain-like motif of CAF-1 is primarily responsible for ASF1a binding.
We next compared CAF-1 p60 binding to ASF1a and ASF1b, using a fragment of CAF-1 p60 missing only the N-terminal WD40 repeats (residues 376–559), since these repeats are neither necessary nor sufficient for ASF1a binding (, left panel
). As shown in , CAF-1 p60(376–559) binds equally well to ASF1aN, ASF1bN and full length ASF1a, consistent with earlier findings that CAF-1 p60 associates with both ASF1a and ASF1b in vivo20
Additional binding experiments using the ASF1aN(D37A) mutant, a mutant which does not bind to HIRA (), were also carried out. These experiments reveal that the ASF1aN(D37A) mutant does not bind to CAF-1 p60 (376–559), nor to the HIRA B-domain, indicating that CAF-1 p60 makes interactions with the ASF1a and ASF1b core domains that are analogous to interactions of the HIRA B-domain with ASF1a.
These data suggest that the HIRA B-domain and the B-domain-like motifs of CAF-1 p60 bind to the ASF1a core domain in a similar and, therefore, mutually exclusive fashion. To directly test this, we purified ASF1aN/HIRA(427–472) and ASF1aN/CAF-1 p60(469–559) complexes and mixed them with increasing concentrations of HIRA peptide (453–467) (0, 1 and 5 mM). As shown in , the HIRA peptide displaces the majority of the CAF-1 p60(469–559) fragment, and moderately displaces the HIRA B-domain (427–472) from ASF1a. An unrelated peptide did not displace either HIRA or CAF-1 p60 at the highest concentration (). This confirms that HIRA and CAF-1 p60 bind mutually exclusively to the N-terminal core of ASF1a.