Structural Plasticity in Human Heterochromatin Protein 1β

As essential components of the molecular machine assembling heterochromatin in eukaryotes, HP1 (Heterochromatin Protein 1) proteins are key regulators of genome function. While several high-resolution structures of the two globular regions of HP1, chromo and chromoshadow domains, in their free form or in complex with recognition-motif peptides are available, less is known about the conformational behavior of the full-length protein. Here, we used NMR spectroscopy in combination with small angle X-ray scattering and dynamic light scattering to characterize the dynamic and structural properties of full-length human HP1β (hHP1β) in solution. We show that the hinge region is highly flexible and enables a largely unrestricted spatial search by the two globular domains for their binding partners. In addition, the binding pockets within the chromo and chromoshadow domains experience internal dynamics that can be useful for the versatile recognition of different binding partners. In particular, we provide evidence for the presence of a distinct structural propensity in free hHP1β that prepares a binding-competent interface for the formation of the intermolecular β-sheet with methylated histone H3. The structural plasticity of hHP1β supports its ability to bind and connect a wide variety of binding partners in epigenetic processes.


Introduction
Proteins of the Heterochromatin Protein 1 (HP1) family are important regulators of chromatin structure and function in almost all eukaryotes. The human genome encodes three HP1 isoforms, hHP1a, hHP1b and hHP1c with distinct sub-nuclear localization and potential activity [1]. HP1a and HP1b are mainly found at heterochromatin sites where they mediate chromatin condensation and gene silencing. HP1c has also been found in euchromatin domains where it seems to be involved in the expression of active genes [2]. The pivotal role of HP1 proteins in genome regulation and their putative connection to the development of cancer [3] have motivated increasing efforts to understand the molecular basis of their biological activity.
HP1b is of particular interest as it is the only isoform essential for viability in mammals [4]. This protein has a multi-domain organization common to the whole HP1 family. A long weakly conserved hinge region links two globular modules, the chromodomain (CD) and the chromoshadow domain (CSD) at the N-and C-terminal sides, respectively. Two additional highly charged regions constitute the N-and C-terminal tails. CD is a ''histone post-translational modification (PTM) reader''. A binding pocket characterized by a conserved aromatic cage selectively discriminates the methylation states of Lys9 in histone H3 [5][6][7]. The trimethylated form (H3me3K9) of this chemical modification is one of the most studied epigenetic marks associated with gene silencing [8]. The NMR structure of CD [9] has revealed a globular shape made of a three-stranded anti-parallel b-sheet packed against a Cterminal a-helix. In the complex, the histone H3 peptide acquires an extended conformation and forms an intermolecular bsandwich with CD [5]. The CSD has a similar fold with the key difference of two a-helices at the C-terminus that constitute an interface for HP1 dimerization [10]. The CSD dimerization interface provides an additional binding platform for diverse protein partners containing a common PXVXL motif [11]. For HP1a, it was shown that the C-terminal tail cooperates with CSD to discriminate the binding among different partners [12]. HP1, via the CSD, can bind proteins from different biological pathways such as transcriptional repression (KAP1) [10,13], chromatin assembly (CAF1) [14], nucleosome remodeling (ATRX) [15], nuclear lamina organization (LBR) [16] and DNA replication (ORC) [17]. The ability of CD and CSD to recruit protein partners from diverse biological networks makes HP1 a powerful molecular connector of different cellular pathways.
The hinge region contains a nuclear localization sequence and has the most variable amino acid sequence among human HP1 isoforms and HP1 from different species. It has been reported to be highly accessible to proteases [9] and it was suggested to be unstructured [10]. Chemical modifications on the linker, especially phosphorylation [18], influence HP1 localization, interaction and function in Drosophila melanogaster. Beyond the basic CD-CSD connection function, the hinge region seems therefore of functional relevance in tuning HP1 activity. Moreover, for some HP1 isoforms, it can bind DNA [19,20]. Recently we showed that interactions of the hinge region and the N-terminal tail with DNA mediate the weak association of hHP1b to unmodified nucleosomes, thus providing an alternative mechanism of chromatin binding besides the specific recognition of methylated histone H3 by the CD [21]. Similarly, the N-terminal tail is weakly conserved and contains residues available for post-translational modifications that can modulate HP1 binding to chromatin [22].
The bifunctional and dimeric nature of HP1 appears to be the key for its biological function. While structures have been deposited for the isolated CD and CSD, no detailed information is available for the full-length protein. In particular, little information about the conformational propensities and dynamics of the hinge region and the long N-and C-terminal tails is available. We therefore used NMR spectroscopy to investigate the structural and dynamical properties of both the non-globular and folded domains in full-length human HP1b. Our study reveals both inter-domain motions and internal dynamics in CD and CSD that can promote complex formation with different binding partners.

hHP1b Populates an Extended Ensemble
To obtain insight into the global conformation of the 184residue full-length hHP1b in solution we investigated its hydrodynamic behaviour. Dynamic light scattering measurements resulted in a well-defined monodisperse peak with a hydrodynamic radius of 4.460.1 nm ( figure 1A). The value is in agreement with the results from pulse field gradient NMR (figure 1B) and indicates that the protein does not assume a compact state in solution.
SAXS experiments were then performed to obtain information about the size and shape of the conformational ensemble populated by hHP1b in solution. For hHP1b at 1.0-5.0 mg/mL concentrations, we obtained a MW of 4064 kDa and an excluded volume of the particle of 8565 nm 3 . The data confirmed that the protein is present in a dimeric state within the range of tested concentrations. Values of the radius of gyration (R g ) and maximal size of the particle (D max ) (4.760.2 nm and 15.560.5 nm, respectively) as well as the long tail of the distance distribution function p(r) (figure 1C, inset) pointed to a relatively elongated shape of hHP1b. To take into account the dynamic nature of hHP1b, SAXS data were also subjected to the ensemble optimization method, which is particularly suited for flexible multi-domain proteins as it accounts for multiple configurations of disordered linkers [23]. Comparison of the R g distribution derived from the optimized ensembles with that obtained from a pool of randomly generated models is shown in figure 1D. The R g distribution of the selected ensemble is nearly as broad as the one from the initial random pool, with the maximum shifted towards longer distances, indicating that the hinge region is highly flexible with a preference for more extended conformations.

CD and CSD do not Form Stable Inter-domain Contacts in Full-length hHP1b
The 1 H-15 N TROSY-HSQC spectrum of 15 N-perdeuterated hHP1b (figure 2A) highlights its multi-domain nature. Besides the two structurally related CD (21-71) and CSD (110-170), the remainder of the protein, which accounts for more than one third of the sequence, has a non-globular character with a high percentage of charged residues. The non-globular nature of the hinge region and the N-and C-terminal tails lead to severe signal overlap between 8 and 8.5 ppm in the 1 H dimension and a large dynamic range of peak intensities. In particular, the clustering of lysine and glutamate residues in the sequence complicates the sequence-specific resonance assignment by conventional tripleresonance techniques, thus precluding the structural characterization of the full-length protein so far. By using automated projection spectroscopy (APSY) experiments on hHP1b (2-185), in combination with the use of protein fragments, we were able to assign most of the hHP1b backbone signals [21].
Analysis of the average 1 H-15 N chemical shift differences between CD in hHP1b (2-185) and the isolated CD (residues 2-79) (figure 2B, upper panel), and between CSD in hHP1b (2-185) and the isolated CSD (residues 107-176) (figure 2B, lower panel), suggested that the structures of both domains are retained in the full-length protein. Moreover, the lack of chemical shift changes except at the termini of the domains excludes significant interdomain contacts between CD and CSD, in line with the conclusions reported by Brasher et al. [10]. The mutual independence of the two domains was further supported by 1 H-15 N residual dipolar coupling (RDC) analysis. Alignment tensors calculated from experimental RDCs and 3D structures had different magnitudes: 9.67 Hz for CD and 29.54 Hz for CSD.

Structural Propensities in the Non-globular Domains
The conformational properties of the hinge region and of the Nand C-terminal tails were addressed by analysis of NMR secondary chemical shifts, that are highly sensitive probes of local conformation [24]. For most residues in the tails and the hinge region the absolute values of Ca secondary chemical shifts were below 0.3 ppm (figure 2C and, for the combined secondary chemical shifts see figure S1), supporting their intrinsically disordered nature. In the hinge region a weak helical tendency expands the a-helix of the CD beyond residue 70 up to 73. In addition, a continuous stretch of small negative Ca secondary shifts in proximity to residue 20 points to a propensity for extended conformations in the N-terminal tail. The extended conformation in this region might be favored by the high density of charged residues and may be functionally relevant for formation of the bsheet sandwich between strand b1 of the CD and the induced bstrand in methylated histone H3. In line with this hypothesis, b1 is N-terminally extended in the dmHP1CD-methylated histone H3 complex [5].

Modular Dynamics of hHP1b
To obtain insight into the dynamic properties of hHP1b, we performed 15 N spin relaxation measurements [25]. In the globular CD and CSD the average R 1 (R 2 ) relaxation rate was 1.2460.11 s 21 (14.8562.62 s 21 ) and 0.7160.11 s 21 (37.0164.54 s 21 ), respectively (figures 3A-B). In addition, average at identical conditions. B. PFG-NMR based diffusion plot of hHP1b. The natural logarithm of the intensity ratio I/I 0 linearly correlates (R = 0.98) with Q, a combined parameter dependent on the gradients strength and delays as defined in [51]. For the intensity ratio, four integrated signals in the 2.4-0.7 ppm region were measured from 32 spectra recorded with increasing gradient strength from 5-75% of the maximum value. Diffusion coefficient values (D) from NMR and DLS experiments were converted into hydrodynamic radius (R h ) values based on the Stokes-Einstein's equation. C. Small angle X-ray scattering profile of hHP1b. The plot displays the decimal logarithm of the scattering intensity as a function of momentum transfer, s. The distance distribution function is displayed in the inset. D. R g distributions from EOM for hHP1b: initial random pool (continuous line) and selected ensembles averaged over 50 independent EOM runs (dashed line). doi:10.1371/journal.pone.0060887.g001 hetNOE values of 0.7060.04 for CD and 0.6460.13 for CSD (figure 3C) indicated that the protein backbone is very rigid in the two domains. From the R 2 /R 1 ratios average t c values of 10.6861.45 ns and 23.4762.41 ns were calculated for the CD and CSD, respectively. Residue-specific t c values in the Cterminal a-helical region of CD were consistently higher than the average t c of the domain (figure 3E), indicating that the global motion of CD is anisotropic. Also CSD shows features of anisotropic global motion: the product of R 1 and R 2 (R 1 R 2 ) efficiently removes the anisotropy that causes a large distribution of R 2 /R 1 [26] (figure S2A).
To characterize the anisotropy of the global motion of CD and CSD within full-length hHP1b, we determined the rotational diffusion tensor of the two domains using the program ROTDIF [27] ( Table 1). The best fit for CD was obtained with the axiallysymmetric diffusion model (Q = 0.35), resulting in average values for t c and anisotropy of 10.1961.18 ns and 2.0260.38, respectively. The orientation of the diffusion tensor of CD is illustrated in figure 3F. The C-terminal helix of CD is aligned nearly parallel to the z-axis of the diffusion tensor, providing a rationale for its large t c values. The CSD data were best fit using the fully-anisotropic diffusion model (Q = 0.37) ( Table 1 and figure 3F) with average t c of 24.5764.95 ns. The anisotropy and rhombicity of the diffusion tensor were 3.5561.41 and 0.3760.13, respectively.
Next we compared the rotational correlation times for the two globular domains in full-length hHP1b to values predicted for the isolated domains using the program HYDROPRO [28]. HY-DROPRO estimated the t c values of the isolated CD and the isolated dimeric CSD as 4 ns and 10 ns, respectively. Thus, rotational correlation times in the isolated domains are more than a factor of two smaller than in full-length hHP1b. The strong increase in t c for the two globular domains in the full protein points to the presence of motional coupling. With the apparent lack of any persistent structure in the intervening hinge region or any stable contact between the two domains, this motional coupling seems to be mainly contributed by hydrodynamic interaction: in the spatial proximity of the other domains, tumbling of each domain is slowed down in comparison with the isolated state as a consequence of a stronger resistance to the accompanied solvent displacement. The presence of hydrodynamic coupling has been demonstrated for several multi-domain proteins, for example a two-domain model protein [29], and appears to be a generic feature of modular proteins with flexible linkers.
According to 15 N spin relaxation rates the backbone outside of CD and CSD is highly mobile (figure 3). The high mobility of these regions precludes the analysis of their dynamic properties through separation of global and internal motions. Therefore, we analyzed 15 N spin relaxation rates by reduced spectral density mapping to describe protein NH vector motions at time scales corresponding to three different frequencies 0, v N and 0.87v H [30] ( figure 3D and figure S3). The N-and C-terminal tails as well as the hinge region showed much smaller J(0) and bigger J(0.87v H ) than the two globular domains, indicating a very slow decay of their spectral density function characteristic of fast tumbling molecules with high internal mobility. The J(0.87v H ) profile demonstrated that the N-terminal tail experiences smaller internal dynamics when compared to the hinge region, with the C-terminal tail being the most flexible part among the disordered domains (figure S3A). A reduced mobility on the pico-to-nanosecond time scale in the N-terminal tail is further supported by an average hetNOE value of 0.1860.08. Interestingly, the N-terminal tail is more rigid at its beginning up to residue L14, where the hetNOE reaches a local minimal value of 0.06, and then starts to rise afterwards (figure 3C).

Internal Dynamics in the Binding Pockets of CD and CSD
Internal dynamics play a key role for molecular recognition [31]. Therefore, we investigated the internal motions in CSD and CD, the two domains that mediate binding of HP1 to a wide variety of protein partners. In CSD, residues E165 to W170 showed the largest 15 N linewidths (figure 4A). Besides a possible local effect due to H171, the observed signal broadening points to slow conformational rearrangements. Importantly, this region is involved in interactions with different proteins, suggesting that this conformational plasticity may support interaction with PXVXL motif and its variants in CSD binding partners.
In CD, which is essential for binding to methylated histone H3 in chromatin, strong signal broadening (figure 4A) and slow chemical exchange (slower than ,100 ms) (figure 4B) was observed for H75, close to the hinge region, and K33, which is located in the short loop between b-strands b1 and b2. The mobility of the loop comprising K33 was further supported by a prominent exchange contribution to the 15 N R 2 relaxation rate and distinct temperature sensitivity of its peak intensity (figure S4). Besides K33, peak intensities of four residue stretches in CD (L27-D28; E56-C60; L63-I64; Q69-S70) showed pronounced temperature sensitivity that points to a change in the flexibility of these regions as a function of temperature ( figure S4C).

N57 to C60 of the CD Populate Binding-competent Conformations Prior to Binding to Methylated Histone H3
How is the structure of CD and CSD in solution affected by the observed conformational dynamics? To address this question, we measured residual dipolar couplings that describe the orientation of internuclear vectors and are therefore highly sensitive probes of structure and dynamics [32]. Experimental 1 H-15 N RDCs of CSD in full-length hHP1b correlated well with values back-calculated from the X-ray structure of CSD (Pearson's correlation coefficient R = 0.97 and dipolar coupling quality factor Q = 0.15) (figure 5A). The fit of experimental RDCs to the X-ray structure of the CD was of much lower quality with R = 0.89 and Q = 0.44. In particular, six RDC values deviated significantly. Their removal improved the quality of the fit to R = 0.98 and Q = 0.18 ( figure 5B). The six RDC values belong to the two residue stretches V32-K33 and N57-C60, which experience a wide range of internal motions ( figure 4 and figure S4).
Inspection of the 3D structure of CD reveals that N57-C60 is part of the intervening region between strand b3 and helix a1 (figures 5B and C). In the X-ray structure of the isolated, unbound CD, a helical turn following the b3-strand has been assigned to residues E56-L58 (PDB code: 3F2U [33]). However, NMR secondary chemical shifts (marked in figure 2C and figure S1A-C) rather point to a propensity for extended conformation in the L58-D59 region. Thus, crystallization may have stabilized the b3-a1 intervening region in a conformation that is weakly represented The average uncertainty threshold is estimated as 0.1 ppm for the nonglobular parts, 0.2 ppm for CD and 0.3 ppm for CSD due to different relaxation properties of these regions. In CD and CSD the presence of segments of continuous positive and negative secondary Ca shifts identifies, respectively, the helix and b-sheet elements in agreement with the secondary structure, schematically shown at the top, as defined in 1AP0 [9] and 1DZ1 [10] PDB files and definition by DSSP [58]. Blue (extended) and green (helical) stripes highlight the additionally identified secondary structure propensities. doi:10.1371/journal.pone.0060887.g002 in solution providing a rationale for why N57-C60 coordinates from the X-ray structure are not compatible with experimental RDCs. Upon interaction with methylated histone H3, residues L58-D59 are stabilized in a well-ordered intermolecular bsandwich, as shown for CD of hHP1a (PDB code: 3FDT [6], displayed in figure 5C in comparison with 3F2U structure), hHP1b (PDB code: 1GUW [7]) and dmHP1 (PDB code: 1KNE [5]). We therefore tested whether our experimental 1 H-15 N RDCs better fit to those back-calculated from the available crystal structures of bound CD (figure 5D-E and figure S1D). A best fit of 43 experimental RDCs spanning V23-Q69 resulted in R = 0.88 and Q = 0.44 when using the PDB of free-CD of hHP1b (PDB code: 3F2U), R = 0.97 and Q = 0.18 with the PDB of bound-CD of hHP1a (PDB code: 3FDT), and R = 0.98 and Q = 0.16 with the PDB of bound-CD of hHP1c (PDB code: 3TZD [34]). The strong improvement in the quality of the RDC fit supports the presence of a binding-competent conformation for N57-C60 in the CD prior to interaction with methylated histone H3, where L58-D59 have already a propensity for more extended conformation. Upon binding, N57-C60 become more rigid as evidenced by an increase of hetNOE values in this region in the complex of hHP1b with the H3K C 9me3 peptide (figure 4C).

Conclusions
Here we showed that hHP1b explores a wide conformational space by populating an extended ensemble. Due to the high flexibility of the hinge region the two globular domains, CD and CSD, remain independent. The absence of direct contacts enables a largely unrestricted spatial search by the two domains for their binding partners. This feature is of primary importance in the activity of HP1 to bridge nearby nucleosomes to form heterochromatin [21,35,36], and to recruit and connect different binding partners belonging to diverse pathways related to chromatin function [37]. In addition, the intrinsic disorder of the hinge region facilitates post-translational modifications by enzymes such as protein kinases [18,38], makes the linker an accessible hold for interaction with nucleic acids [18,20,21,39] and increases the exposure of the nuclear localization sequence for proper localization [37]. Our data moreover reveal that, despite the high flexibility of the hinge region, the rotational diffusion of both CD and CSD is evidently slowed down by the presence of other domains within the full-length protein. Thus, hHP1b provides an example of how hydrodynamic interaction alters the tumbling of domains within flexible modular proteins.
Both CD and CSD have distinct structural and motional properties in regions important for molecular recognition. In CSD, strong signal broadening between E165 and W170 points to the presence of slow conformational rearrangements that can be useful for the versatile recognition of the PXVXL motif and its variants [40]. Moreover, the N57-C60 region, which is stabilized in an intermolecular b-sheet upon interaction of the CD with methylated histone H3, has a propensity for the binding-competent conformation already in free hHP1b. The propensity for extended structure is enforced upon interaction with histone peptide to complete the b-barrel architecture of the domain [41]. Interestingly, structures of the same CD bound to different peptides, such as CD of HP1c with a histone1.4 peptide and CD of HP1c with a histone-lysine N-methyltransferase peptide [34], show different backbone geometries in this region, indicating that a certain degree of conformational plasticity could better accommodate different binding partners. In summary, the structural plasticity of hHP1b promotes its activity in binding and connecting a variety of proteins related to epigenetic events.
All samples were finally prepared in 20 mM sodium phosphate (pH 6.5), 50 mM NaCl, 2 mM DTT and 0.02% NaN 3 and filtered before the experiments.
The H3K C 9me3 1-15 peptide was obtained from a synthetic peptide H3(1-15)K9C where a Cys residue replaced the Lys at position 9. This modification allowed the site-specific installation of a tri-methyl lysine analog by Cys-alkylation reaction using (2bromoethyl)-trimethylammonium bromide (Sigma). Details of the alkylation reaction were described previously [21,42]. Diffusion tensor parameters for the different tumbling models, obtained from experimental 15 N spin-relaxation data through ROTDIF, are listed: the overall rotational correlation time t c ; D xx , D yy and D zz are the principal values of the diffusion tensor; Q is the quality factor defined as in [27]; P defines the probability that an improvement in the fit when a more complex model is applied has occurred by chance. The best model for CD and CSD is marked in bold. The little improvement in the fit with the more complex fully-anisotropic model was not statistically significant for CD. doi:10.1371/journal.pone.0060887.t001

Dynamic Light Scattering
Dynamic light scattering measurements were performed on a Wyatt DynaPro Titan instrument (Wyatt Technology, California) at 303 K on a sample containing 0.1 mM hHP1b.

SAXS Experiments
Synchrotron radiation X-ray scattering data were acquired on the EMBL X33 beamline at the DORIS III storage ring, DESY, in Hamburg [43]. Experiments were carried out at 283 K, with protein concentrations of 1.0, 2.0 and 5.0 mg/mL. A pixel detector PILATUS 1 M (DECTRIS, Switzerland) at sampledetector distance 2.7 m and wavelength l = 0.15 nm, covering the momentum transfer range 0.12,s,4.9 nm 21 (s~4p sin (h)=l where 2h is the scattering angle), was employed. Data were processed with the ATSAS program package [44]. For each measurement, four 30 sec exposures were compared to check for radiation damage. No radiation effects were observed. The data were averaged after normalization to the intensity of the incident beam. The signal of the buffer was subtracted and the difference data were extrapolated to zero solute concentration by standard procedures.
Data analysis was performed with the program PRIMUS [45]. The forward scattering I(0) and the radius of gyration R g were obtained using the Guinier approximation, assuming that at very small angles (s,1.3/R g ) the intensity is represented as I (s)~I(0) exp ({(1=3)(R g s) 2 ). These parameters were also computed from the entire scattering patterns with the program GNOM [46], giving the distance distribution function p(r) and the maximum particle dimension D max . The MW was obtained comparing the forward scattering to that from reference solutions of bovine serum albumin (66 kDa). Possible flexibility of hHP1b was assessed by the ensemble optimization method (EOM) [23], which allows for coexistence of different conformations contributing to the experimental scattering pattern. These conformers were selected by a genetic algorithm from a pool containing 10 5 randomly generated models. Genetic algorithms were employed to find the subsets of the conformers that fit the experimental data best. The obtained subsets were analysed to yield the R g distributions in the optimal ensembles.
Secondary chemical shifts were calculated based on the random coil chemical shifts predicted by the Neighbor Corrected Structural Propensity Calculator [49] and corrected for the 2 H isotope shift. The 4,4-dimethyl-4-silapentane-1-sulfonic acid (0.0 ppm) was used for chemical shift referencing. Consensus chemical shift index (CSI) values were obtained using the RCI webserver [50].
Pulse field gradient stimulated-echo diffusion experiments were performed at 303 K on a sample containing 0.12 mM of hHP1b. Q was defined according to [51]. Diffusion gradient length (little delta) and diffusion delay (big delta) were set to 3 ms and 200 ms, respectively. Gradient calibration was achieved by measuring the diffusion of residual HDO in 99.8% D 2 O at 298 K.  15 N longitudinal relaxation rates (R 1 ) were measured using relaxation delays of 8, 30, 60, 100, 180, 320, 500, 800 and 1200 ms. 15 N transverse relaxation rates (''total R 2 '') were measured using relaxation delays of 8,16,24,34,52,86,120,180 and 240 ms. 15 N longitudinal relaxation rates in the rotating frame (R 1r ) were measured in a near-resonance mode with relaxation delays of 20, 40, 60, 80, 100, 120, 140 and 180 ms and a spin-lock field strength of 2.5 kHz. R 1 , R 2 and R 1r relaxation rates were determined from the best single exponential fit to the experimental intensity data. Effective R 2 rates were derived from the relation: R 2~( R 1r {R 1 cos 2 h) sin 2 h, where h~tan {1 (n 1 =V), n 1 is 15 N spin-lock field strength (in Hz) and V is the resonance offset from the spin-lock carrier (Hz). R ex values were determined as the difference between the ''total R 2 '' rates and the effective R 2 rates derived from R 1r values. Steady-state 1 H-15 N heteronuclear nuclear Overhauser enhancement (NOE) was measured with a total recycle delay of 10 s. NOE values were calculated by the ratio of the peak intensities between saturated and reference spectra. With the reduced spectral density mapping, the relaxation rates R 1 and effective R 2 and hetNOEs were transformed to spectral densities at zero frequency J(0), at 15 N frequency J(v N ) and at the effective 1 H frequency J(0.87v H ) [30]. The theoretical relation between J(v) and J(0) was obtained with the assumption of a single Lorentzian motion, as J (v)~J(0) . (1z6:25v 2 J 2 (0) ). Outliers are marked in orange. In A and B, residues affected by overlap or with insufficient signal-to-noise ratio were excluded from the analysis. C. Alignment between free CD of hHP1b (PDB code: 3F2U) and bound CD of hHP1a (PDB code: 3FDT). The b3-a1 intervening region that includes the N57-C60 residues (outliers in the RDCs analysis) is shown in red for free CD and in blue for bound CD. The histone peptide of 3FDT PDB is coloured in light blue. D. For rotational diffusion analysis the program ROTDIF7 [27] and the crystal structures of the isolated CD (PDB code: 3F2U) and CSD (PDB code: chain A, 2FMM) were used. For each of the two domains, only residues within the main secondary structural elements were included in the analysis. Three diffusion models, isotropic, axially-symmetric and fully-anisotropic were utilized for calculation of rotational diffusion tensors, and the F-test was applied to evaluate if the improvement of the fit by the more complex model was statistically significant. 500 Monte-Carlo calculations were performed for uncertainty analysis. The R 2 used in this analysis was the exchange-free R 2 0 derived from the 15 N-1 H dipole-dipole/ 15 N chemical shift anisotropy cross-correlated relaxation rates (g xy ) according to R 0 2~k g xy z1:3s, where s~(NOE{1)R 1 c N =c H . The results with the R 1r -based effective R 2 were consistent with these results, but had larger uncertainty range for the CSD domain. R 1r -based R 2 g xy , i.e. k (kappa), was highly similar for the two domains: 1.3960.05 for CD and 1.4160.14 for CSD. These values are consistent with a 15 N CSA magnitude of 2162 ppm and an angle of 19.6u between 15 N CSA and internuclear N-H vector, in line with the recent reports [52]. g xy rates (figure S2B) were measured using relaxation delays (2D) of 6, 8, 10, 20 and 60 ms, and determined by signal intensity analysis according to: I A =I B~t anh (2Dg xy ), where I A and I B are the signal intensities obtained with pulse schemes A and B in [53], which separately address relaxation of 15 N downfield and upfield doublet components. R 1r -based relaxation dispersion experiments for K33 were performed on a sample containing 0.48 mM of 15 N-labelled CD  at 303 K and a proton Larmor frequency of 600 MHz. R 1r rates were obtained at 15 N spin-lock field strengths of 200, 600, 1000 and 1500 Hz centered on the 15 N resonance of K33, using six relaxation time delays between 5 and 190 ms.
One bond 1 H-15 N coupling constants of protein backbone were obtained using a TROSY-HSQC interleaved experiment recorded on a 900 MHz NMR spectrometer equipped with a cryoprobe. The sample contained 0.2 mM of 15 N-perdeuterated hHP1b. The temperature was 293 K. Partial alignment was achieved using a dilute liquid crystalline phase of 5% C12E5/ hexanol (Sigma) [54] resulting in 16 Hz of quadrupolar splitting. RDC data were analyzed using the PALES software [55]. Alignment tensors calculated from experimental RDCs and 3D structures (PDB codes: 3F2U and 2FMM, chain A) had magnitudes of 9.67 Hz for CD and 29.54 Hz for CSD with rhombicities of 0.09 and 0.12, respectively. NMR data were processed with NMRPipe [56] and analyzed with Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco). OriginH and Wolfram MathematicaH were used for mathematical and graphical analyses. CD and CSD PDBs were visualized with MolMol [57] and PyMOL (The PyMOL Molecular Graphics System, Version 1.1r1, LLC). Figure S1 Secondary chemical shifts and RDCs analysis. A, B. Combined Ca+CO (A) and Ca-Cb (B) secondary chemical shifts as a function of residue number. The average uncertainty threshold is estimated 0.1 ppm for the non-globular parts, 0.2 ppm for CD and 0.3 ppm for CSD. Blue (extended) and green (helical) stripes highlight the additionally identified secondary structure propensities. The Ca-Cb secondary chemical shifts have the advantage that they are not affected by any possible imperfection in 13