Goats from the Zagros Mountains may represent the earliest examples of goat herding.
Image credit: The Central Zagros Project.
Edited by Tony Pawson, Mt. Sinai Hospital, Toronto, ON, Canada, and approved October 9, 2009 (received for review July 29, 2009)
Abstract
Haspin, a nuclear and chromosome-associated serine/threonine (S/T) kinase, is responsible for mitotic phosphorylation of Thr-3 of histone H3. Haspin bears recognizable similarity to the eukaryotic protein kinase (ePK) fold, but its sequence is highly divergent and there is therefore considerable interest in its structural organization. We report the 2.15-Å crystal structure of the kinase domain of human Haspin. The ePK fold of Haspin contains an array of insertions and deletions. The structure illustrates how Haspin escapes the classical activation scheme of most other kinases. The αC helix, which bears a conserved glutamate that is essential for catalysis, adopts its final active conformation within the small lobe of the kinase. It is sandwiched between an α-helical insertion that precedes the kinase domain, and the activation segment, which adopts an unprecedented conformation. The activation segment, which does not contain phosphorylatable residues, packs against an unusually structured αEF helix. Significantly extruded from the core of the fold, it forms an extensive plateau, hosting several residues implicated in substrate binding. Overall, the structure of the Haspin kinase domain reveals an active conformation that is poised for substrate recognition and phosphorylation in the absence of external regulators.
Protein kinases are essential players in cell physiology and their deregulation is implicated in several pathological conditions (1). The eukaryotic protein kinase (ePK) fold, whose prototype was revealed by the structure of the cAMP-dependent protein kinase (2), consists of a small N-terminal lobe (N-lobe), containing five β-strands and the conserved αC helix, and a larger, predominantly α-helical C-terminal lobe (C-lobe). The catalytic cleft, containing an ATP-binding site and a substrate-binding site, is located at the interface between the N- and C-lobes. The activity of protein kinases is controlled by a multitude of regulatory mechanisms (3–5). Regulation by phosphorylation of one or more key residues within the activation segment is typical. The activation segment, located in the C-lobe, consists of a metal-binding site (DFG motif), a short β-strand (β9), the activation loop, and the P+1 loop (6). The phosphate moiety stabilizes the activation segment in a conformation suitable for substrate binding (6). In contrast, the activation segment in the inactive unphosphorylated state is usually largely unstructured and unfit to recognize substrates (4, 6).
Several atypical kinases exist in the human genome (7). Atypical kinases are homologous to the ePK family and are therefore likely to adopt the bilobed ePK fold, but they lack at least one of the conserved catalytic residues and might therefore be enzymatically inactive or alternatively their activation mechanism might differ significantly from that of typical kinases (7). Haspin/Gsg2 (haploid germ cell-specific nuclear protein kinase/germ cell-specific gene-2) is an atypical kinase with weak similarity to the ePK profile (7). Haspin-family kinases share similar domain architecture, with a kinase domain occupying the C-terminal part of the molecule and a low complexity and possibly partially unfolded N-terminal region with a reduced degree of sequence identity within the family (Fig. 1A) (8, 9). Haspin is a nuclear protein that associates with chromosomes to phosphorylate threonine 3 of histone H3 (P-Thr-3H3) preferentially within mitotic centromeres (10–12). This modification has been recently linked to the activation of the Aurora B kinase, a crucial regulator of mitotic progression (13), but previous studies had failed to establish a convincing relationship between Haspin and Aurora B activity (10). In agreement with a mitotic role of Haspin, its knockdown by RNAi causes an accumulation of cells with chromosome cohesion and alignment problems and a metaphase arrest (10, 14).
The structure of Haspin is unknown. Here, we describe the structure of the catalytic domain of human Haspin and discuss and probe its unusual mechanism of activation.
Results and Discussion
Haspin Has High Affinity for Its Substrates.
We bacterially expressed and purified several fragments of the catalytic domain of Haspin. A proteolytically resistant fragment, encompassing residues 452–798 (Haspin452–798), was chosen for an in vitro kinase assay with histone H3 as substrate. This segment also undergoes significant autophosphorylation (see SI Results and Fig. S1 A and B). Kinetic analysis of the phosphorylation reactions employing a NADH-coupled photometric assay revealed that the KM of Haspin452–798 for ATP is 180 μM (Fig. 1B). Previously, it has been reported that full-length Haspin has a KM for histone H3 of 98 nM (15). Although limitations in sensitivity of our assay prevented us from measuring a KM for histone H3, we were able to examine the binding of Haspin452–798 to an immobilized substrate, the N-terminal segment of histone H3 (H31–50-GST), with surface plasmon resonance (SPR). The Haspin kinase domain bound to the immobilized histone H3 with an observed Kd of 0.7 μM (Fig. 1C).
Fig. 1.
Haspin452–798 kinase domain catalytic parameters. (A) A scheme of human Haspin is shown below a disorder probably plot calculated with PrDOS (24). The red dotted line indicates 50% probability. (B Upper) NADH oxidation was monitored as a function of time, converted to nanomoles of ATP consumed and plotted against time (minutes) at different ATP concentrations. (Lower) Velocities were plotted against ATP concentration. (C Upper) BIAcore sensorgrams for the binding of Haspin452–798 to H31–50-GST immobilized on a glutathione-coated chip. Recombinant Haspin452–798 was applied to H31–50 at various concentrations. (Lower) Analysis of equilibrium dissociation constants, with response units at equilibrium (RU eq) plotted as a function of Haspin concentration. RU eq is derived from the steady-state plateau of the association phases of the sensorgrams. Equilibrium dissociation constants were determined with BIAevaluation 4.0.1 (Biacore).
Overall Description of the Haspin Catalytic Domain.
We determined the structure of Haspin452–798 by the single anomalous dispersion (SAD) method from crystals of a selenomethionine-containing derivative (Table 1). The structure reveals the classical bilobate ePK fold, with N- and C-lobes, and a catalytic cleft between them containing residues involved in catalysis (Fig. 2). Despite overall low sequence identity, most critical ePK residues implicated in Mg2+ and nucleotide binding, and additional residues required for catalysis, are conserved in Haspin. On the other hand, the structure reveals at least four main Haspin-specific features. First, the N-lobe is entirely buried under an additional layer created by an N-terminal extension and two conserved insertions (described below). Second, the overall organization of the activation segment and of its surroundings is unconventional and contributes to creating an unusual substrate-binding site. Third, there is a conserved 20-residue insertion between the β7 and β8 loop. Fourth, a deletion in the large lobe removes the αG helix, which in other ePKs occupies a prominent position on the front face of the domain.
View this table:
Table 1.
Data collection and refinement statistics
Fig. 2.
Overall structure of Haspin452–798. (A) Multiple sequence alignment of Haspin-like kinases. Secondary structure elements and degree of conservation are indicated. Three insertions described in the text are marked by a red bar. (B) Cartoon representation of two rotated views of Haspin452–798 showing N-terminal extension (orange), β3-αC loop (green), αC (blue), αC′ (dark red), activation segment (red), and β7′-β7′ hairpin (yellow). (C) The additional layer on the N-lobe is visualized after a ≈45° rotation from the view in B. Side chains of residues making critical interactions as described in the text are displayed. (D) p21 activated kinase (Pak1) (pdb:1YHV) was colored as in B to emphasize the presence of an N-terminal extension analogous to that observed in Haspin (17). Note that the additional insertions are absent in Pak1.
Reorganization of the N-Lobe.
The core of the N-lobe of Haspin consists of a twisted 5-stranded anti-parallel β-sheet and the αC helix (Fig. 2B). A specific feature of Haspin is that the core of the N-lobe is entirely buried under an additional layer built from three distinct structural elements. The first element is an N-terminal extension (residues 452–487) that precedes the glycine-rich loop (β1–β2). The first part of the extension folds as a α helix (αA, residues 452–467). It surmounts the αC helix and contributes to its stabilization (see below). Before connecting to the β1 strand, the extension meanders irregularly on the surface at the far end of the N-lobe, forming two additional short helical segments (αA′ and αA″). Conserved residues in this region, including Gln-469, Phe-475, and Leu-477, contribute to its stabilization with hydrogen bonds (H-bonds) and hydrophobic interactions (Fig. 2C).
Two additional elements are inserted between β3 and αC (insertion 1, residues 515–528) and between β4 and β5 (insertion 2, residues 567–598). Insertion 1 forms an irregular but rigid structure that replaces the short αB helix often observed in other ePK family members such as PKA (Fig. 2C). Insertion 2 is also rigid. Its first part (residues 571–584) folds as an α helix (named αC′) and is locked in place by interactions with the ceiling of the core of the N-lobe mediated by the side chains of Tyr-569 and Pro-570 (Fig. 2C). The side chain of Trp-577 on the αC′ helix buries a tripartite interaction involving the underlying side chains of Lys-489, Glu-492, and Glu-497 in the Gly-rich loop (Fig. 2C). A charged interaction analogous to the Lys-489–Glu-497 interaction is important for catalysis in several ePK family members, probably due to stabilization of the Gly-rich loop (16). Being surmounted by insertion 2, the Gly-rich loop is further stabilized and is therefore highly ordered (average temperature factor of 48.5 Å2 against an average temperature factor of 49.2 Å2 for the entire model). The second part of insertion 2 meanders on the surface of the N-lobe, contributing with the side chains of Phe-593 and Phe-594 to a hydrophobic core that also includes Val-521 from insertion 1 and several aliphatic and aromatic side chains from the top surface of the β-sheet of the N-lobe (Fig. 2C). Together, the N-terminal extension and insertions 1 and 2 form a continuous system and they reinforce each other through additional interactions, most notably between the side chains of Gln-526 and Gln-598.
Haspin Adopts the Active Conformation.
The structure of Haspin452–798 likely depicts the catalytically active conformation. The side chains of Lys-511 and Glu-535 (in the β3-strand and αC helix, respectively), two conserved residues whose side-chains are almost invariably in close contact in active kinases but not in inactive kinases, are only 2.7 Å away (Fig. 3A), indicating that the αC helix (on which Glu-535 is located), which is usually a very mobile element of kinases, has adopted a position compatible with catalysis.
Fig. 3.
Role of αA helix in Haspin activation. (A) A representation of the hydrophobic environment around αC (blue) reveals stabilization by the activation segment (red) and by the αA helix in the N-terminal extension (orange). (B) Different Haspin variants were separated by SDS/PAGE and stained with Coomassie. (C) Indicated Haspin variants were tested to determine the ability to phosphorylate histone H3 in vitro in the presence of radiolabeled ATP. The reactions were then separated by SDS/PAGE and visualized by autoradiography.
Two distinct sets of interactions, the first with residues in the αA helix (N-terminal extension) and the second with a hydrophobic cluster from the activation segment, contribute to the stabilization of the active conformation of the αC helix (Fig. 3A). To dissect the role of the αA helix, we tested the relative kinase activity of N-terminal variants of the Haspin kinase domain. In thermal denaturation experiments, all variants showed similar protein stability (Fig. S2). Haspin406–798 and Haspin452–798, both containing the αA helix, efficiently phosphorylated histone H3. Conversely, constructs lacking the αA helix (Haspin468–798) were significantly less active (Fig. 3 B and C). A similar decrease in activity was observed when Val-463, a residue at the hydrophobic interface between the αA and αC helices, was replaced with Asn (Haspin452–798 V463N) (Fig. 3). In contrast, abolishing the salt bridge between Arg-695 and Glu-466 (Haspin452–798 E466A), which connects the activation segment to the αA helix, did not affect significantly the ability of Haspin to phosphorylate histone H3, indicating that this feature is not essential for kinase activity (Fig. 3). In an accompanying paper, Knapp and colleagues report the independent structure determination of a construct of human Haspin (465–798) lacking the αA helix (PDB ID 2VUW). Haspin452–798 and Haspin465–798 are structurally very similar (Fig. S3), suggesting that the differences in activity, reported in Fig. 3 B and C, are associated with modest structural changes (see SI Discussion).
Other kinases contain an αA helix preceding the small lobe. In p21-activated kinase 1 (Pak1, PDB ID code 1yhv) (17), whose structure is rather closely related to Haspin's (overall rmsd for 198 Cα atoms is 3 Å, Fig. 2D), the N-terminal αA helix packs against the surface formed by the αC helix and the β4–β5 strands. An equivalent helix (αL16, residues 338–353) is also present in the structure of the extracellular signal-regulated kinase 2 kinase domain (18). In other kinases, such as the CDKs, the αC helix is pushed into the active conformation by equivalent interactions with cyclins and cyclin-like proteins (19).
Conformation of the Activation Segment.
The second block of interactions keeping the Haspin αC in place implicates residues in the activation segment. The tip of the activation segment (residues 692–700) forms a β-hairpin (β9–β9′) (Figs. 2–4). Leu-693 and Val-700 in the β9–β9′ hairpin contact a hydrophobic cluster of the αC helix containing Pro-534, Ile-537, and Ile-538 (Fig. 3A). Overall, the Haspin activation segment is shorter than in classical ePK folds, such as PKAs, and its sequence and conformation are uncommon (Fig. 4A). For instance, the segment starts with an unusual 687-Asp-Tyr-Thr-689 (DYT) motif, rather than with the usual Asp-Phe-Gly (DFG) motif (Figs. 2A and 4A). However, the side-chain conformation of the Asp and Tyr residues of Haspin are similar to the ones observed in typical DFG motifs in active kinases. Additionally, the Ala-Pro-Glu (APE) motif that normally closes the segment is absent (Fig. 4A).
Fig. 4.
Conformation of Haspin's activation segment. (A) Multiple sequence alignment of activation segments in the indicated kinases. The secondary structure of the loop in Haspin452–798 is Above the alignment, whereas the usual succession of structural elements in prototype kinases such as PKA is shown Below the alignment. (B) The activation segment and αEF helix in Haspin (Left) and PKA (Right). The αE and αF helices are shown as a reference as they superimpose very well in the two structures. The activation segment of Haspin is extruded into solvent due to several aromatic residues contributed by the segment itself and by the αEF helix. (C) Arg-648 in the HRD motif of Haspin (Left) coordinates the main chain of the αEF–αF loop, Right, Below the activation segment. In the phospho-CDK2/cyclin A complex (Right), the equivalent arginine is implicated in activation loop stabilization by binding to the activation loop phosphate (25).
After the β9–β9′ hairpin, the chain is extruded into solvent due to the presence of the bulky αEF helix (residues 716–729), which runs almost parallel to the αF helix (Fig. 4B). The chain then turns again and connects to αF. This region, which roughly corresponds to the P+1 loop and the aEF helix of other kinases (see Fig. 4A), is completely reorganized. Several conserved aromatic and hydrophobic residues, including Val-704, Leu-710, Phe-711, Phe-719, Tyr-722, and Trp-733 contribute to a hydrophobic core that, together with additional hydrophilic interactions with the core of the kinase, stabilizes the unusual conformation of this region. Remarkably, the side chain of Arg-648 in the His-Arg-Asp (HRD) motif, which in those kinases containing the HRD motif is usually implicated in the stabilization of the phosphorylated activation segment (see Fig. 4C for an example), in Haspin is H-bonded to the main chain carbonyl groups of Trp-733 and Glu-735, thus contributing to shape the αEF–αF loop (Fig. 4C).
Additional Structural Features.
A characteristic element of Haspin is the presence of a 20-residue insertion extending the β7–β8 loop (insertion 3). Insertion 3 contains two additional β-strands, β7′ and β7″, engaging in a β-hairpin. The hairpin extends toward the back of the catalytic domain, where it makes contacts with the αC–β4 loop, mainly through main-chain interactions. This might explain why the sequence of insertion 3 is poorly conserved within the Haspin family (Fig. 2 A and B). In typical kinases such as PKA (2), the αG helix lies at the front of the C-lobe, beside the activation loop (Fig. 4B), an excellent position to influence substrate recognition and specificity in protein kinases (20, 21). In Haspin, the αG helix is absent, and the chain progresses directly from the end of the αF helix to the beginning of the αH helix (Fig. 2A).
Haspin Binds and Phosphorylates Nucleosomes in Vitro.
In vitro, Haspin452–798 phosphorylates histone H3. It also phosphorylates a single band, which we identify as histone H3, within purified chicken erythrocyte nucleosomes (Fig. S4). Because Haspin binds a peptide substrate with high affinity (Fig. 1), we asked whether Haspin physically interacts with nucleosomes. When loaded onto a Superdex-200 size-exclusion chromatography (SEC) column, Haspin452–798 and nucleosomes eluted as expected for globular structures of 35 kDa and 280 kDa, respectively (Fig. S4). When Haspin452–798 and nucleosomes were combined stoichiometrically and then analyzed by SEC, Haspin452–798 coeluted with nucleosomes in a stoichiometric or near-stoichiometric complex, suggesting high affinity binding (Fig. 5A). When Haspin452–798 and nucleosomes were combined with Mg2+ and ATP, we only observed a partial release of Haspin from nucleosomes, possibly suggesting that Haspin retains the ability to interact with a phosphorylated substrate, albeit at lower affinity (Fig. 5B). To corroborate this idea, we prephosphorylated H31–50-GST (P-H31–50-GST) (Fig. 5C). Haspin452–798 (and a catalytically impaired version of the same kinase) bound to H31–30-GST, H31–50-GST, and P-H31–50-GST (Fig. 5D). Using SPR, we determined that Haspin452–798 binds to P-H31–50-GST with a Kd of 7 μM, a 10-fold decrease in binding affinity relative to the nonphosphorylated substrate (Figs. 5E and 6E).
Fig. 5.
Haspin binds nucleosomes in vitro. (A) Haspin kinase and nucleosomes were mixed, separated by SEC, and analyzed by SDS/PAGE. A stoichiometric amount of Haspin coelutes with nucleosomes, indicative of complex formation. (B) Haspin kinase and nucleosomes were mixed with Mg2+ and ATP, and later separated by SEC and analyzed by SDS/PAGE. (C) H31–50-GST was phosphorylated in vitro in a cold kinase assay with Haspin452–798 (see Methods). Incorporation of phosphate on Thr-3 is demonstrated by an anti-P-Thr-3 monoclonal antibody. The preparation was subjected to a further step of phosphorylation in the presence of γ-[32]ATP. No radioactivity was incorporated in the prephosphorylated sample, indicating that it has been phosphorylated to completion in the first round with cold ATP. (D) GST, H31–30-GST, H31–50-GST, or P-H31–50-GST on GSH beads were incubated with the Haspin452–798 or Haspin452–798-D687A (KD). After a washing step, SDS/PAGE and Coomassie staining were used to visualize bound species. (E) Binding of Haspin452–798 to immobilized H31–50-GST. Response units at equilibrium (RUeq) were measured on a Biacore device and plotted as a function of protein-kinase concentration. Kd values were calculated by measuring association (ka) and dissociation (ka) rate constants from Biacore sensorgrams. Kd values were calculated as Kd = kd/ka (see Fig. 6E).
Fig. 6.
The substrate-binding pocket. (A) Electrostatic potential mapped onto the molecular surface of the Haspin kinase domain. Red: negative; blue: positive. (B) Sequence conservation was mapped onto the molecular surface. (C) Analysis of equilibrium dissociation constants for Haspin kinase wild type (WT) and indicated mutants. Response units at equilibrium (RUeq) are plotted as a function of protein-kinase concentration. (D) Surface representation of the residues tested in this study for substrate binding properties. (E) Kd, ka, and kd values for several Biacore binding experiments involving Haspin452–798 and its mutants as mobile ligands, and H31–50-GST or P-H31–50-GST as an immobilized receptor.
Identification of Residues Implicated in Substrate Recognition.
Besides autocatalysis (Fig. S1), the only known substrate of Haspin is Thr-3 of histone H3. Thr-3 lies within the conserved sequence 1-Ala-Arg-Thr-Lys-Gln-5 (10). Arg-2 and Lys-4 are both required for efficient H3 binding and phosphorylation by Haspin (10). As expected for a positively charged substrate, there is a negative charge distribution around the catalytic cleft (Fig. 6A), which is lined by residues that are highly conserved in the Haspin family (Fig. 6B). Because we have been unable to cocrystallize Haspin with a peptide substrate, we applied mutational analysis to identify Haspin residues involved in H3 substrate recognition. Wild type (WT), catalytically inactive (D687A), and different point mutants of Haspin (Fig. S5A) were tested for their ability to phosphorylate histone H3 in vitro (Fig. S5B). Because Haspin has a low KM (15) and a low Kd for histone H3 (Fig. 1), we used histone H3 at a concentration of 100 nM in our reactions to maximize the likelihood of observing an effect of the mutations on kinase activity. In these qualitative experiments, substitution of Glu-492, His-651, Asp-707, Asp-709, Gly-713, and Asp-716 with other residues markedly affected histone H3 phosphorylation (Fig. S5B). To gain a quantitative understanding of the effects of these mutations, we investigated their effect on the enzymatic parameters of Haspin. We determined catalytic efficiency (kcat) and KM for ATP in a kinase assay in which the H3 substrate was fixed at a concentration of 5 μM. Three mutants, D611L, H651A, and D716L, also had reduced kcat, suggesting either a role in catalysis or reduced stability of the enzyme. Besides a reduced kcat, D716L also had an increased KM for ATP (Fig. S5C). At least four mutants, E492A, D707L, D709N, and G713F, displayed normal kcat and KM for ATP (Fig. S5) but reduced affinity for H3 (Fig. 6 C–E), suggesting that they are important for substrate recognition. Consistently, all four mutants were severely impaired in H3 phosphorylation when assayed at nonsaturating concentrations of substrate (Fig. S5B). The mutated residues line the catalytic pocket of Haspin (Fig. 6D). His-651, identified above for having a reduced catalytic efficiency, also displayed a severely reduced affinity for the substrate (Fig. 6 C–E). Thus, mutations in this residue appear to affect both substrate recognition and catalytic efficiency. This result is consistent with the position of His-651, whose side chain flanks the side chain of the catalytic Asp-649 (Fig. 6D).
Conclusions
The structure of the atypical kinase domain of Haspin reveals an unprecedented mechanism of activation. An extensive set of hydrophobic interactions impart to the αC helix and to the activation segment a conformation compatible with catalysis, even in the absence of activation loop phosphorylation, suggesting that Haspin is a constitutively active kinase. Haspin is active after removal of at least four prominent autophosphorylation sites and is therefore unlikely to be regulated by phosphorylation.
The amino-terminal region of Haspin (1–450) is a low-complexity sequence that is unlikely to fold as a stable globular domain. It is possible that the specific sequences in the Haspin N-terminal domain regulate the access of the kinase domain to its substrates, in a manner analogous to that recently described for the spindle checkpoint kinase Bub1, another atypical kinase (22).
We have also shown that the Haspin kinase domain binds to nucleosomes with high-affinity. The low Kd for histone H3 shown here and suggested previously by KM measurements (15) may account, at least in part, for the high-affinity interaction revealed by our binding studies. Indeed, we found a 10-fold reduction in the affinity of Haspin for the phosphorylated form of H3, indicating that the substrate-binding site is implicated in binding. Nucleosomes might be expected to influence the catalytic activity of the kinase domain to deliver catalytic activity directly onto nucleosome-bound substrate. In the future, it will also be interesting to address the question of how the modification of histone tails, and in particular modifications of residue in the vicinity of Thr-3, influence substrate recognition. As the phosphorylation of Thr-3H3 is restricted to the centromere (10), it is plausible that specific centromeric and pericentromeric chromatin modifications (such as Lys-4H3 methylation and acetylation or Lys-9H3 methylation) influence the recognition of Thr-3H3 by Haspin (23).
Methods
Plasmids and Protein Expression.
A cDNA segment encoding Haspin452–798 was subcloned in a modified pGEX-6P vector (GE Healthcare) as a C-terminal fusion to GST, with an intervening PreScission protease site. Point mutants were created with the QuikChange kit (Stratagene). All constructs were sequence verified. Escherichia coli BL21(DE3) cells harboring vectors expressing Haspin452–798 were grown at 37 °C to an OD600 of 0.7–0.9. IPTG (0.15 mM) was added and the culture grown at 23–25 °C for 12–16 h. The GST–Haspin452–798 chimera was affinity purified on GSH beads using standard protocols. After a cleavage reaction with PreScission protease, the supernatant was loaded onto a Resource Q column (GE Healthcare) equilibrated with IE buffer [50 mM Tris (pH 7.6), 1 mM DTT]. Haspin452–798 was eluted from the column with HIE buffer [50 mM Tris (pH 7.6), 2M NaCl and 1 mM DTT] with a liner gradient, concentrated, and loaded onto a Superdex 200 SEC column equilibrated with SEC buffer [25 mM Tris HCl (pH 7.6), 150 mM NaCl, 1 mM DTT]. Haspin452–798 was finally concentrated to 12 mg/mL. Additional constructs are described in SI Methods.
Crystallization and Structure Determination.
Haspin452–798 was crystallized by sitting drop vapor diffusion. Diffraction-quality crystals were obtained against a reservoir containing 7% PEG3350 and 100 mM NaAcetate pH 5.3. A selenomethionine (SeMet) derivative crystallized under similar conditions. Diffraction data from native and derivative crystals were collected at beamlines ID29 (SeMet) and ID14–1 (native) (ESRF-Grenoble) (Table 1). The computational aspects of structure determination are described in SI Methods.
Acknowledgments
We thank the staff at the European Synchroton Radiation Facility (Grenoble) for help in data collection, V. Cecatiello for assistance in crystallization, Dr. N. Avanzi (Nerviano Medical Science) for help with the NADH-coupled assay, and the Musacchio group and Stefan Knapp for discussions. F.V. is an EMBO long-term postdoctoral fellow. This work was funded by grants from Fondazione Cariplo (to A. Musacchio and A. Mattevi), Fondo di Investimento per la Ricerca di Base (FIRB) (to A. Musacchio and A. Mattevi), and Italian Ministry of Health through the PIO RO Strategici 7/07 (to A. Musacchio). A. Musacchio holds an advanced investigator grant of the European Research Council (KINCON).
Footnotes
- 1To whom correspondence may be addressed. E-mail: fabrizio.villa{at}ifom-ieo-campus.it or andrea.musacchio{at}ifom-ieo-campus.it
Author contributions: F.V. and A. Musacchio designed research; F.V. performed research; P.C., M.T., F.F., and A. Mattevi contributed new reagents/analytic tools; P.C., A.d.M., A. Mattevi, and A. Musacchio analyzed data; and A. Musacchio wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2WB8).
This article contains supporting information online at www.pnas.org/cgi/content/full/0908485106/DCSupplemental.
Freely available online through the PNAS open access option.
References
Crystal structure of the catalytic domain of Haspin, an atypical kinase implicated in chromatin organization
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