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Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme

2001; Springer Nature; Volume: 20; Issue: 19 Linguagem: Inglês

10.1093/emboj/20.19.5320

1460-2075

Karsten Niefind, Bárbara Guerra, Inessa Ermakowa, Olaf‐Georg Issinger,

Plant biochemistry and biosynthesis

Article1 October 2001free access Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme Karsten Niefind Corresponding Author Karsten Niefind Universität zu Köln, Institut für Biochemie, Zülpicher Straße 47, D-50674 Köln, Germany Search for more papers by this author Barbara Guerra Barbara Guerra Syddansk Universitet, Institut for Biokemi og Molekylær Biologi, Campusvej 55, DK-5230 Odense, Denmark Search for more papers by this author Inessa Ermakowa Inessa Ermakowa Universität zu Köln, Institut für Biochemie, Zülpicher Straße 47, D-50674 Köln, Germany Search for more papers by this author Olaf-Georg Issinger Olaf-Georg Issinger Syddansk Universitet, Institut for Biokemi og Molekylær Biologi, Campusvej 55, DK-5230 Odense, Denmark Search for more papers by this author Karsten Niefind Corresponding Author Karsten Niefind Universität zu Köln, Institut für Biochemie, Zülpicher Straße 47, D-50674 Köln, Germany Search for more papers by this author Barbara Guerra Barbara Guerra Syddansk Universitet, Institut for Biokemi og Molekylær Biologi, Campusvej 55, DK-5230 Odense, Denmark Search for more papers by this author Inessa Ermakowa Inessa Ermakowa Universität zu Köln, Institut für Biochemie, Zülpicher Straße 47, D-50674 Köln, Germany Search for more papers by this author Olaf-Georg Issinger Olaf-Georg Issinger Syddansk Universitet, Institut for Biokemi og Molekylær Biologi, Campusvej 55, DK-5230 Odense, Denmark Search for more papers by this author Author Information Karsten Niefind 1, Barbara Guerra2, Inessa Ermakowa1 and Olaf-Georg Issinger2 1Universität zu Köln, Institut für Biochemie, Zülpicher Straße 47, D-50674 Köln, Germany 2Syddansk Universitet, Institut for Biokemi og Molekylær Biologi, Campusvej 55, DK-5230 Odense, Denmark *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5320-5331https://doi.org/10.1093/emboj/20.19.5320 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 Å resolution (Protein Data Bank code: 1JWH). In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure. Introduction Protein kinase CK2 (casein kinase 2) is one of the most unspecific eukaryotic protein kinases: first, >160 in vitro protein substrates of CK2 have been described to date (Pinna and Meggio, 1997); secondly, CK2 shows the rare ability to use either ATP or GTP as phosphoryl donor (dual-co-substrate specificity) (Niefind et al., 1999); thirdly, although known as a serine/threonine kinase for several decades, the capability of CK2 also to phosphorylate tyrosine has been reported repeatedly in recent years (Chardot et al., 1995; Wilson et al., 1997; Marin et al., 1999); and fourthly, CK2 activity was found not only with the natural cofactor Mg2+ but also with other divalent cations such as Mn2+ and Co2+ (Gatica et al., 1993). These biochemical properties and its biomedical significance (Guerra and Issinger, 1999) make CK2 a favourite research subject. The enzyme has been found in all eukaryotic cells investigated so far and is highly conserved in evolution, indicating its critical function in cellular life. Within the phylogenetic tree of the protein kinases the catalytic core of CK2 belongs to the CMCG group (Hanks and Hunter, 1995) meaning that its nearest neighbours are key regulatory enzymes, including the cyclin-dependent kinases (CDK) or the mitogen-activated protein kinases. CK2 is essential for the viability of a cell (Padmanabha et al., 1990), and overexpression of its catalytic subunit is correlated with lymphoma development in transgenic mice (Seldin and Leder, 1995). Not least due to its unspecific activity profile and its wide occurrence in tissues and cell compartments, the exact cellular functions of CK2 remain elusive. The metabolic roles of the >160 in vitro substrates of CK2 identified so far (Pinna and Meggio, 1997) have led to many hypotheses about the involvement of CK2 in carcinogenesis, viral tumorigenesis, transcriptional control, apoptosis, cell cycle, signal transduction and other key biological processes (Guerra and Issinger, 1999). It is generally accepted that CK2 plays an important role in cell proliferation and embryogenesis. In parallel with its functional diversity CK2 also presents a complex picture concerning its quaternary structure. In vivo it exists mainly as a holoenzyme composed of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Two isoforms of the catalytic subunit (CK2α and CK2α′) have been found in human and many other sources and CK2 complexes of α2β2, αα′β2 and α′2β2 stoichiometry can occur (Chester et al., 1995). Moreover there is increasing evidence that the isolated subunits can exist in vivo under certain circumstances and possibly have specific functions (Pinna and Meggio, 1997). In vitro the CK2 holoenzyme forms spontaneously from the individual subunits by a self-assembly mechanism mediated by dimerization of the two CK2β chains (Graham and Litchfield, 2000). The formation of higher oligomeric states of CK2 was also observed in vitro (Glover, 1986). At the level of tertiary structure the current knowledge about CK2 is restricted to the individual subunits. Crystal structures have been published for recombinant maize CK2α (rmCK2α; Niefind et al., 1998) and for recombinant and C-terminally truncated human CK2β (rhCK2βΔ; Chantalat et al., 1999), while for the CK2 holoenzyme only a theoretical model derived from the crystal structure of maize CK2α in complex with a short peptide of human CK2β exists (Battistutta et al., 2000). This model suggests a direct contact of the two CK2α subunits within the CK2 complex and is therefore in conflict with yeast two-hybrid system studies showing that CK2α molecules are able to bind to CK2β but not to each other (Gietz et al., 1995; Boldyreff et al., 1996). To clarify this conflict and to rationalize some of the specific biochemical properties of the CK2 holoenzyme on a structural level we describe here for the first time the crystal structure of a complete CK2 tetramer. For this work we used recombinant human CK2. Human CK2α is normally 391 amino acids long (Figure 1A) and has a molecular mass of 45.1 kDa. During purification a 5 kDa peptide was spontaneously cleaved from the catalytic subunit leaving a degraded form of ∼40 kDa (rhCK2αΔ; Niefind et al., 2000), which is around the typical size of CK2α subunits from various sources. The resulting stable CK2 holoenzyme comprising two molecules of rhCK2αΔ and two molecules of recombinant human CK2β (rhCK2β) is fully active with several typical CK2 substrates (Niefind et al., 2000). It is called rhCK2Δ throughout this paper. Whether this partial degradation of human CK2α occurs also in natural host cells and whether the cleaved peptide has a biological function is not known to date. Figure 1.Sequence overview of human CK2α (A) and CK2β (B). Small characters in the sequence indicate residues that are truncated or disordered in both copies of the chains and are therefore left out in the final rhCK2αΔ structure. Positions at which the final 2Fo − Fc electron density is badly defined are printed in italics. The contact residues are assigned with black bars. The α/β contacts normally occur twice in the rhCK2Δ complex; if a certain residue was detected as a contact partner only in one chain it is marked with a grey bar instead of a black one. For human A-Raf and Xenopus laevis c-Mos partial sequences are included in (A). Download figure Download PowerPoint Results and discussion Structure determination The rhCK2Δ structure was solved with crystals grown in the presence of Mg2+ ions and the non-hydrolysable ATP analogue adenylyl imidodiphosphate (AMPPNP). The crystals belong to space group P63 with lattice constants a = b = 176.0 Å, c = 93.7 Å. The asymmetric unit of the crystals contains one rhCK2Δ tetramer and has a solvent content of 61%. We hereafter designate the two rhCK2αΔ chains as A1 and A2 and the two rhCK2β chains as B1 and B2. The crystals were of limited X-ray diffraction quality. The best resolution obtained with a rotating copper anode was 6 Å. At a synchrotron we measured a complete data set to 3.1 Å resolution (Table I), merging data from two crystals. The single diffraction frames showed a significant anisotropy, which was also detected by the program TRUNCATE (CCP4, 1994) for the complete data set. A considerable internal disorder in the crystals was indicated by high mosaicities of the two crystals (1.3 and 1.4°) and an overall B-factor of 92.2 Å2 calculated by TRUNCATE from a Wilson plot. Table 1. Crystallographic analysis Characteristic data of the rhCK2Δ synchrotron data set Space group P63 Lattice constants a = b = 176.0 Å, c = 93.7 Å No. of crystals used 2 Temperature of data collection 100 K Resolution range 59.3–3.1 Å No. of observations 268 848 No. of rejections 6073 No. of independent reflections 29 935 Wilson-plot B-factor 92.2 Å2 Multiplicity 8.8 Multiplicity for last shell (3.2–3.1 Å) 6.0 Average of (I/σI) 16.7 Average of (I/σI) for last shell (3.2–3.1 Å) 2.6 Completeness for whole range 98.4% Completeness for last shell (3.2–3.1 Å) 90.4% Rsym for whole range 9.6% Rsym for last shell (3.2–3.1 Å) 42.0% Overview of the final structure model Resolution range included in refinement 3.1–60.0 Å Rfree (4% of all reflections)a 33.8% Rwork (96% of all reflections)a 26.7% Real space R-valuea 5.3% Average B-factor 91.0 Åb R.m.s.ds for bondsa 0.009 Å R.m.s.ds for anglesa 1.5° Quality of Ramachandran plot % residues in most favouredb regions 69.5 % residues in additional allowedb regions 28.1 % residues in generously allowedb regions 2.2 % residues in disallowedb regions 0.2 Cross-validated sigmaa estimate of mean coordinate errora 1.04 Å Cross-validated Luzzati estimate of mean coordinate errora 0.67 Å Rsym = ΣhΣj|Ih,j − |/ΣhΣjIh,j, where Ih,j is the intensity of the jth observation of unique reflection h, and is the mean intensity of that reflection. Reflection intensities related by Friedel symmetric were merged in this data set. a Calculated with CNS (Brünger et al., 1998). b According to PROCHECK (CCP4, 1994). The structure was solved by a combination of molecular replacement and phase refinement techniques (see Materials and methods) and refined with all reflections from 59.3 to 3.1 Å to a final model with acceptable stereochemical parameters (Table I). A Ramachandran graph for each subunit is shown in Supplementary figure 1, available at The EMBO Journal Online. Quality of the structure Most parts of rhCK2Δ are defined by good electron density (see Figure 2 for examples), while in some sections the quality of the density is reduced as indicated by increased real space R-factors (Supplementary figure 2). Finally there are regions lacking any ordered electron density. Among these, as indicated in Figure 1, the N-terminus of B2 and the zone Asn206 to Arg215 of B1 and B2 were left out from the final model whereas the zone Asp55 to Asn65 of B1 was modelled as a copy of its equivalent in B2. Figure 2.Electron densities in selected regions of rhCK2Δ. All maps apart from that covering the AMPPNP molecule in (D) are sigma-weighted 2Fo − Fc electron densities contoured with a 1σ cutoff. The red map in (D) around AMPPNP is an Fo − Fc omit density (4σ cutoff) calculated after a 5000 K simulated annealing run excluding the AMPPNP coordinates. (A) Stereo figure of the CK2β tail as part of the β/β contact. The two rhCK2β subunits are distinguished by different colours (red and blue) for the electron densities and the labels. His165 and Met169 of rhCK2βΔ (Chantalat et al., 1999) and some corresponding water molecules displaced by the formation of the β/β contact are drawn in black. (B) Stereo figure of the hydrophobic core of the α/β contact. The colour coding for electron densities and labels is black for the CK2β tail with Tyr188 as the central residue of the whole core, red for the body of the second rhCK2β chain and blue for the participating rhCK2αΔ subunit. (C) The N-terminal segment of rhCK2αΔ attached to the activation segment (yellow). For comparison the backbone courses of both segments in rmCK2α (Niefind et al., 1999; PDB code: 1DAW) after superimposing on rhCK2αΔ are drawn in black. Further colour coding for activation segments after three-dimensional fits: red for the partially active CDK2 in complex with a cyclin A fragment (Jeffrey et al., 1995; PDB code: 1FIN) and violet for the inactive CDK2 in isolated form (De Bondt et al., 1993; PDB code: 1HCL). (D) Stereo figure of AMPPNP bound to the active site of rhCK2Δ chain A1. To compare the γ-phosphate positions, AMPPNP as bound to rmCK2α (Niefind et al., 1999) is drawn in black after superimposition of the protein matrices. Download figure Download PowerPoint Some parts of the chains missing in the initial models, for instance the C- and N-termini of A1 and A2 or the functionally critical zone around Tyr188 of B1 and B2, were added during the refinement process. Model building at 3.1 Å resolution and with model-biased phases is difficult and susceptible to errors. We cannot exclude such errors, which will become apparent with higher-resolution diffraction data, but in view of the final electron densities we are nevertheless confident that we have identified the correct structure from Gly3 to Pro6 of A1 and A2, from Arg333 to Ser337 of A1 and from Pro176 to Gly189 of B1 and B2. In some other parts (C-terminus of A2, N-terminus and zone Asp55 to Asn65 of B2, zone Phe190 to Ser205 of B1 and B2) the rough trace of the main chain was clear enough, but the conformations of peptide groups and side chains remain questionable. Within such a zone, namely from Met195 to Gln200 of rhCK2β, we modelled an α-helix (αG; Figures 1B and 3D) in agreement with a secondary structure prediction (Korn et al., 1999) and with circular dichroism spectroscopic data, indicating that the CK2 complex formation is accompanied by an increase in the α-helical content of the protomers (Issinger et al., 1992). Figure 3.Various aspects of the rhCK2Δ structure. (A and B) Overall shape of rhCK2Δ in a view perpendicular to the local C2 axis (A) and along this axis (B). The two rhCK2β chains are drawn in blue and red, the two rhCK2αΔ subunits in yellow and grey. (C) Structural overview of rhCK2αΔ chain A1 with bound AMPPNP and interdomain flexibility. The hinge axis and the bending residues of the domain closure motion as detected by DYNDOM (CCP4, 1994) are included. To illustrate the interdomain flexibility, the N-terminal domains of rmCK2α (PDB code: 1DAW) and of rhCK2αΔ chain A2 are shown in yellow and black, respectively, after three-dimensional alignment of the corresponding C-terminal domains. (D) Structural overview of rhCK2β. The human CK2β peptide bound to rmCK2α (black) was taken from PDB file: 1DS5 (Battistutta et al., 2000) after superimposition of the corresponding CK2α subunits. (E) Intersubunit flexibility at the α/β contact. Subunit A1 is drawn with yellow colour for the C-terminal domain and grey for the N-terminal domain. Subunit B1 bound to A1 by an α/β contact is sketched in red. Subunit B2 is shown in blue after a three-dimensional fit of the N-terminal domain of subunit A2 (not drawn) on that of A1. Download figure Download PowerPoint In the active site of subunit A1 a bound AMPPNP molecule—but no accompanying Mg2+ ions—was detected while A2 was free from a co-substrate analogue. Several peaks of residual electron density were filled with water molecules and phosphate ions in accordance with a phosphate concentration of ∼0.2 M in the crystallization drops (Niefind et al., 2000). In agreement with rhCK2βΔ (Chantalat et al., 1999) a zinc ion was found in both regulatory subunits. With 91.0 Å2 the average B-factor of the structure is relatively high, but it corresponds well with the B-factor obtained from a Wilson plot (92.2 Å2; Table I). Furthermore the R-factors are relatively large (Table I). While crystal twinning was excluded as a reason, this is likely to be a consequence of the conservative refinement strategy and of the disorder in the crystals, which is indicated by zones of diffuse electron densities and by the drawbacks of the data set mentioned above (low resolution, anisotropy, high mosaicity, high overall B-factor). Overall architecture of rhCK2Δ and its subunits Shape of the complex. The rhCK2Δ complex has the shape of a butterfly in a view from above perpendicular to the molecular 2-fold axis (Figure 3A). Its approximate dimensions in the directions of the principal axes are 155 × 90 × 66 Å. The second of these axes coincides with the molecular dyad. Figure 3A and B shows that the central building block of the rhCK2Δ complex is the rhCK2β dimer bridging the space between both rhCK2αΔ chains. This happens in such a way that each of the two rhCK2β monomers touches both rhCK2αΔ subunits, which in contrast make no contact with each other. In fact the centres of mass of the two rhCK2αΔ molecules are 98.5 Å, their active sites ∼80 Å and their nearest atoms 32.4 Å distant from one another (Figure 3A and B). This central result confirms former findings based on the yeast two-hybrid system (Gietz et al., 1995; Boldyreff et al., 1996) and is consistent with the monomeric state generally found for isolated CK2α subunits. In contrast it disproves the theoretical CK2 holoenzyme model of Battistutta et al. (2000) in which the two catalytic subunits are postulated to be in close contact and the active sites only 13 Å apart from one another. The shape of rhCK2Δ in the view of Figure 3B resembles the quaternary structure models that Zhao et al. (1998) derived for the holoenzyme of cAMP-dependent protein kinase (CAPK) from neutron small angle scattering experiments. In these models, as in rhCK2Δ, the catalytic C subunits do not touch and have a centre-of-mass distance of as much as 122 Å, while the R2C2 complex is formed by the central dimer of two regulatory R subunits. Hence the organization of the rhCK2Δ complex presented here may also be representative for CAPK and other protein kinases. Structure of the catalytic subunits. Both rhCK2αΔ subunits show the typical bilobal architecture of the catalytic core of eukaryotic protein kinases with a β-sheet-rich N-terminal domain, an α-helical C-terminal domain and the active site between them (Figure 3C). Structural superpositions of the two rhCK2αΔ subunits and of isolated rmCK2α (Niefind et al., 1998), including virtually all Cα atoms, lead to root mean square deviations (r.m.s.ds) of ∼0.8 Å (Table II). These values demonstrate the overall similarity of the superimposed structures, although a closer inspection discloses an interdomain flexibility, which is discussed below. Table 2. Minimal r.m.s.ds after three-dimensional fits of Cα atoms Catalytic subunits rhCK2Δ, chain A1a rhCK2Δ, chain A2a rhCK2Δ, chain A1 0.0 Å (337) 0.92 Å (331) rhCK2Δ, chain A2 0.92 Å (331) 0.0 Å (336) rmCK2α (1DAWb) 0.91 Å (327) 0.76 Å (326) Regulatory subunits rhCK2Δ, chain B1a rhCK2Δ, chain B2a rhCK2Δ, chain B1 0.0 Å (200) 0.25 Å (199) rhCK2Δ, chain B2 0.25 Å (199) 0.0 Å (204) Isolated CK2β, chain A (1QF8b) 0.82 Å (165) 0.85 Å (165) Isolated CK2β, chain B (1QF8b) 0.67 Å (163) 0.76 Å (163) Dimers rhCK2Δ, CK2β dimer Isolated CK2β, full dimer (1QF8b) 0.83 Å (328) Δ rhCK2, CK2β dimer 0.83 Å (328) a The values in brackets are the numbers of matched Cα atoms. b RCSB Protein Data Bank code. One of the most conspicuous structural features of rmCK2α, a strong attachment of the N-terminal segment to the activation segment from Asp175 to Glu201 (Niefind et al., 1998), is also found in the catalytic subunits of rhCK2Δ and hence is not affected by rhCK2β (Figure 2C). This observation disproves the hypothesis of Sarno et al. (1998) that CK2β and the N-terminal segment of CK2α are competitive for attachment to the activation segment. The conservation of the activation segment conformation in isolated and complex-bound CK2α is in remarkable contrast to CDK2, where binding of a cyclin A fragment changes the conformation of the activation segment dramatically and brings CDK2 to a partially active conformation (Figure 2C; Jeffrey et al., 1995). In this way cyclin A is a molecular switch for CDK2 activity similar to the R-subunit in the case of CAPK. In contrast CK2α is catalytically active both in isolated and in complex-bound form. As an isolated molecule it has a basal activity, but this is elevated for most substrates significantly by CK2β, for example, for the synthetic peptide RRDDDSDDD by a factor of four (Boldyreff et al., 1994). Calmodulin, however, serves as a much better substrate for CK2α than for the CK2 holoenzyme. More complicated still, these stimulatory and inhibitory effects of CK2β on CK2α strongly depend on the salt concentration and on the presence of effector molecules such as polyamines or polybasic peptides (Pinna and Meggio, 1997). Taken together, CK2β is an environment- and substrate-dependent modulator of CK2α activity rather than an on–off switch (Boldyreff et al., 1994). This constitutive activity fully agrees with the structural similarity between isolated and complex-bound CK2α. Structure of the regulatory subunits. The r.m.s.ds in Table II show that the structure of the CK2β body changes only a little as a consequence of the formation of the rhCK2Δ complex, and further that the two rhCK2β molecules of the complex are more similar to one another than the two rhCK2αΔ subunits. This difference is certainly due to the intense interactions between the two rhCK2β chains imposing constraints in favour of structural similarity, whereas the rhCK2αΔ subunits are not in contact with one another and hence much more free for conformational variations. Each of the rhCK2β molecules can be divided into a body comprising an α-helical N-terminal domain and a Zn2+-containing domain on the one hand and a C-terminal tail on the other (Figure 3D). While the CK2β body has been characterized by Chantalat et al. (1999), the C-terminal tail of CK2β is a new structural element. It points away from the body, forms a 90° knee with a β-hairpin loop with Tyr188 at its top and becomes more and more disordered afterwards (Figure 2A). The importance of this loop is indicated by the fact that the sequence zone R186LYGFKI192 is highly conserved. Gly189 and Phe190 are present in all CK2β sequences currently known, while at position 188, apart from tyrosine, phenylalanine also occurs. The CK2β tail has no contact with the body of its own monomer (Figure 3D) but is stabilized mainly by hydrophobic interactions with the second rhCK2β (Figure 2A) and with one of the rhCK2αΔ subunits (Figure 2B). These interactions are described below. The conformation of the CK2β tail is partially consistent with the 3.1 Å resolution structure of rmCK2α in complex with a peptide comprising positions 181–203 of human CK2β (Battistutta et al., 2000; PDB code 1DS5). These authors correctly identified the β-hairpin loop (Figure 3D) and its binding site at CK2α. However, the structure of the rmCK2α-bound peptide deviates more from rhCK2Δ the larger the distances are from the hairpin loop (Figure 3D). This drawback and the absence of any structural overlap with the rhCK2βΔ structure of Chantalat et al. (1999) are certainly the reasons for the wrong architecture of the CK2 holoenzyme model derived by Battistutta et al. (2000). This failure demonstrates the limits of a peptide-based approach for the detection of the architecture of an oligomeric protein complex. Subunit interactions Overview and designations. The two principal types of protein–protein complexes are homocomplexes between identical subunits, which are usually permanent and evolutionary optimized, and heterocomplexes between non-identical chains, which can be also permanent but are quite often non-obligatory, that is they are made and broken according to the environment and external factors. The size of a protein interface allows an estimation of the permanent or non-obligatory character of a protein–protein contact (Jones and Thornton, 1996). The rhCK2Δ complex is a mixture of both complex types. (i) An rhCK2β homodimer formed by an interface that we call ‘β/β contact’ further on is at the centre of rhCK2Δ around the molecular 2-fold axis (Figure 3A and B). (ii) There are two ‘α/β contacts’ between the rhCK2β dimer and either rhCK2αΔ chain, respectively. Each of these contacts is heterotrimeric, meaning that the interface is composed of one rhCK2αΔ chain and both rhCK2β subunits (Figure 3A and B). To distinguish the individual contributions we use the designations ‘α/β body contact’ and ‘α/β tail contact’ further on. An ‘α/β body contact’ connects an rhCK2αΔ subunit and the body of an rhCK2β monomer, while an ‘α/β tail contact’ connects an rhCK2αΔ subunit and the tail of an rhCK2β monomer. The β/β contact. An r.m.s.d. of 0.83 Å between isolated and complex-bound CK2β dimers (Table II) demonstrates that the whole CK2β dimer arrangement is conserved during CK2 complex formation. Consequently the basic principles of the β/β contact in the isolated CK2β dimer (Chantalat et al., 1999) are also valid for the CK2β bodies in rhCK2Δ. (i) The interface is formed by the Zn2+-binding motif (Figure 1B). (ii) The interface is mainly hydrophobic with a central area composed of Pro110, Val112, Leu124, Val143 and the hydrophobic parts of Tyr113 and Tyr144. (iii) Well defined intersubunit salt bridges (Arg111 with Asp142; Figure 2A) and hydrogen bonds (Pro110 O with Thr145 N; Val143 O with Val112 N) provide a further stabilization. Chantalat et al. (1999) argue about the fact that the buried surface at the β/β contact in dimers of C-terminally truncated CK2β amounts to only 543 Å2, which is less than a third of the typical value for protomers of that size (Jones and Thornton, 1996). In rhCK2Δ this paradox is solved because here the β/β interface, with 1766 Å2 per subunit, is more than three times as big. The reason for this increase is that in rhCK2Δ not only the CK2β body contributes to the β/β contact but in an extensive manner so does the tail (Figures 1B and 2A). For example, a novel hydrogen bond is formed from the peptide oxygen of Arg186 in one rhCK2β subunit to the side chain of Thr161 of the other (Figure 2B). And above all in the centre of the new contact region a hydrophobic cluster of Met141, Val143, Tyr136, Phe168, His165 and Met169 from one chain and Phe183, Pro185, Leu187, Ile192 and Met195 from the other forms. Figure 2A illustrates how the side chains of His165 and Met169 change their conformations to participate in this hydrophobic zone and how water molecules found in isolated rhCK2βΔ dimers (Chantalat et al., 1999) are displaced from it. In particular His165 manages in this way to become a member of the aromatic core of the hydrophobic cluster together with Tyr136 and Phe168 from its own and Phe183 from the other subunit (Figure 2A). The remarkable increase in the β/β contact by the CK2β tail, however, requires a specific conformation of this tail, which also depends—as discussed below—on its participation in the α/β contact and hence on the existence of the complete tetramer. In contrast, for isolated CK2β the region Val170 to Ala180 at the beginning of the tail attenuates the dimerization (Boldyreff et al., 1996). This demonstrates the synergistic character of the CK2β tail: it causes aggregation and stability problems in isolated CK2β and was therefore removed for its structure determination (Chantalat et al., 1999), but within the CK2 tetramer it stabilizes the β/β and α/β contacts. The compact nature of the rhCK2β dimer and the extended β/β contact compared with isolated CK2β dimers fully agree with the central role of CK2β dimer formation as the initial step of CK2 holoenzyme assembly (Graham and Litchfield, 2000). The α/β contacts. The contacts of either catalytic subunit with the CK2β dimer are restricted to the N-terminal lobe of the common core (Figure 1A) typical for all eukaryotic protein kinases (Hanks and Hunter, 1995). More precisely parts of the outer surface of the central β-sheet within the N-terminal domain form the interface with the rhCK2β dimer. Minor contact zones in rhCK2αΔ are found at strand β3, at the subsequent loop and further at the loop connecting the strands β4 and β5. The main contact region is one of the most important for catalytic activity, namely the strands β1 and β2 and the glycine-rich ATP-binding loop in between (Figu