Unexpected CK2ß-antagonistic functionality of bisubstrate inhibitors targeting protein kinase CK2.

Protein kinase CK2, a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) attached to a homodimer of regulatory subunits (CK2ß), is a target for drug development for cancer therapy. Here, we describe the tetraiodobenzimidazole derivative ARC-3140, a bisubstrate inhibitor addressing the ATP site and the substrate-binding site of CK2 with extraordinary affinity (Ki = 84 pM). In a crystal structure of ARC-3140 in complex with CK2α, three copies of the inhibitor are visible, one of them at the CK2ß interface of CK2α. Subsequent interaction studies based on microscale thermophoresis and fluorescence anisotropy changes revealed a significant impact of ARC-3140 and of its tetrabromo equivalent ARC-1502 on the CK2α/CK2ß interaction. A structural inspection revealed that ARC-3140, unlike CK2ß antagonists described so far, interferes with both sub-interfaces of the bipartite CK2α/CK2ß interaction. Thus, ARC-3140 is a lead for the further development of highly effective compounds perturbating the quaternary structure of the CK2α2ß2 holoenzyme.

Arabidopsis immunity regulator EDS1 in a PAD4/SAG101-unbound form is a monomer with an inherently inactive conformation.

In plant innate immunity, enhanced disease susceptibility 1 (EDS1) integrates all pathogen-induced signals transmitted by TIR-type NLR receptors. Driven by an N-terminal α/ß-hydrolase-fold domain with a protruding interaction helix, EDS1 assembles with two homologs, phytoalexin-deficient 4 (PAD4) and senescence-associated gene 101 (SAG101). The resulting heterodimers are critical for EDS1 function and structurally well characterized. Here, we resolve solution and crystal structures of unbound Arabidopsis thaliana EDS1 (AtEDS1) using nanobodies for crystallization. These structures, together with gel filtration and immunoprecipitation data, show that PAD4/SAG101-unbound AtEDS1 is stable as a monomer and does not form the homodimers recorded in public databases. Its PAD4/SAG101 anchoring helix is disordered unless engaged in protein/protein interactions. As in the complex with SAG101, monomeric AtEDS1 has a substrate-inaccessible esterase triad with a blocked oxyanion hole and without space for a covalent acyl intermediate. These new structures suggest that the AtEDS1 monomer represents an inactive or pre-activated ground state.

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2ß interaction.

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2ß in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α(1-335) and the fluorescent probe CF-Ahx-Pc as a CK2ß analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α(1-335). Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2ß. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α(1-335) complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α(1-335) were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2ß, with an IC50 value of 92µM.

Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET(2).

Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET(2) pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP(2). This BRET(2) sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET(2) sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate ß-arrestin2, decreased the BRET(2) signal by up to 50%. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50%. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET(2) sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination.

Quinate dehydrogenase (QDH) catalyzes the reversible oxidation of quinate to 3-dehydroquinate by nicotineamide adenine dinucleotide (NADH) and is involved in the catabolic quinate metabolism required for the degradation of lignin. The enzyme is a member of the family of shikimate/quinate dehydrogenases (SDH/QDH) occurring in bacteria and plants. We characterized the dual-substrate quinate/shikimate dehydrogenase (QSDH) from Corynebacterium glutamicum (CglQSDH) kinetically and revealed a clear substrate preference of CglQSDH for quinate compared with shikimate both at the pH optimum and in a physiological pH range, which is a remarkable contrast to closely related SDH/QDH enzymes. With respect to the cosubstrate, CglQSDH is strictly NAD(H) dependent. These substrate and cosubstrate profiles correlate well with the details of three atomic resolution crystal structures of CglQSDH in different functional states we report here: with bound NAD+ (binary complex) and as ternary complexes with NADH plus either shikimate or quinate. The CglQSDH-NADH-quinate structure is the first complex structure of any member of the SDH/QDH family with quinate. Based on this novel structural information and systematic sequence and structure comparisons with closely related enzymes, we can explain the strict NAD(H) dependency of CglQSDH as well as its discrimination between shikimate and quinate.

A splicing variant of EDS1 from Vitis vinifera forms homodimers but no heterodimers with PAD4.

Enhanced Disease Susceptibility 1 (EDS1), a key component of microbe-triggered immunity and effector-triggered immunity in most higher plants, forms functional heterodimeric complexes with its homologs Phytoalexin Deficient 4 (PAD4) or Senescence-associated Gene 101 (SAG101). Here, the crystal structure of VvEDS1Nterm , the N-terminal domain of EDS1 from Vitis vinifera, is reported, representing the first structure of an EDS1 entity beyond the model plant Arabidopsis thaliana. VvEDS1Nterm has an α/ß-hydrolase fold, is similar to the N-terminal domain of A. thaliana EDS1 and forms stable homodimers in solution as well as in crystals. These VvEDS1Nterm homodimers are spatially incompatible with heterodimers with PAD4 or SAG101, they explain why VvEDS1Nterm does not interact with V. vinifera PAD4 according to gel filtration, and they serve as a guide to develop a plausible, albeit experimentally not verified model of full-length EDS1. VvEDS1Nterm is a splicing variant comprising two of three exons of the VvEDS1 gene. It originates from a naturally occurring mRNA, in which the first of two introns was removed while the second one containing a stop codon close to the exon/intron border was retained. This is a potential case of intron retention and the first report of this phenomenon in the context of EDS1. Its biological significance has not yet been clarified, nor has the question if a VvEDS1Nterm protein with a specific function can occur under physiological conditions.

Analyzing the interactome of human CK2ß in prostate carcinoma cells reveals HSP70-1 and Rho guanin nucleotide exchange factor 12 as novel interaction partners.

CK2ß is the non-catalytic modulating part of the S/T-protein kinase CK2. However, the overall function of CK2ß is poorly understood. Here, we report on the identification of 38 new interaction partners of the human CK2ß from lysates of DU145 prostate cancer cells using photo-crosslinking and mass spectrometry, whereby HSP70-1 was identified with high abundance. The KD value of its interaction with CK2ß was determined as 0.57 µM by microscale thermophoresis, this being the first time, to our knowledge, that a KD value of CK2ß with another protein than CK2α or CK2α' was quantified. Phosphorylation studies excluded HSP70-1 as a substrate or activity modulator of CK2, suggesting a CK2 activity independent interaction of HSP70-1 with CK2ß. Co-immunoprecipitation experiments in three different cancer cell lines confirmed the interaction of HSP70-1 with CK2ß in vivo. A second identified CK2ß interaction partner was Rho guanin nucleotide exchange factor 12, indicating an involvement of CK2ß in the Rho-GTPase signal pathway, described here for the first time to our knowledge. This points to a role of CK2ß in the interaction network affecting the organization of the cytoskeleton.

Low-density crystal packing of human protein kinase CK2 catalytic subunit in complex with resorufin or other ligands: a tool to study the unique hinge-region plasticity of the enzyme without packing bias.

A low-resolution structure of the catalytic subunit CK2α of human protein kinase CK2 (formerly known as casein kinase 2) in complex with the ATP-competitive inhibitor resorufin is presented. The structure supplements previous human CK2α structures in which the interdomain hinge/helix αD region adopts a closed conformation correlating to a canonically established catalytic spine as is typical for eukaryotic protein kinases. In the corresponding crystal packing the hinge/helix αD region is nearly unaffected by crystal contacts, so that largely unbiased conformational adaptions are possible. This is documented by published human CK2α structures with the same crystal packing but with an open hinge/helix αD region, one of which has been redetermined here with a higher symmetry. An overview of all published human CK2α crystal packings serves as the basis for a discussion of the factors that determine whether the open or the closed hinge/helix αD conformation is adopted. Lyotropic salts in crystallization support the closed conformation, in which the Phe121 side chain complements the hydrophobic catalytic spine ensemble. Consequently, genuine ligand effects on the hinge/helix αD conformation can be best studied under moderate salt conditions. Ligands that stabilize either the open or the closed conformation by hydrogen bonds are known, but a general rule is not yet apparent.

A subnanomolar fluorescent probe for protein kinase CK2 interaction studies.

Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α.

Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2αLys198Arg.

Specific de novo mutations in the CSNK2A1 gene, which encodes CK2α, the catalytic subunit of protein kinase CK2, are considered as causative for the Okur-Chung neurodevelopmental syndrome (OCNDS). OCNDS is a rare congenital disease with a high phenotypic diversity ranging from neurodevelopmental disabilities to multi-systemic problems and characteristic facial features. A frequent OCNDS mutation is the exchange of Lys198 to Arg at the center of CK2α's P+1 loop, a key element of substrate recognition. According to preliminary data recently made available, this mutation causes a significant shift of the substrate specificity of the enzyme. We expressed the CK2αLys198Arg recombinantly and characterized it biophysically and structurally. Using isothermal titration calorimetry (ITC), fluorescence quenching and differential scanning fluorimetry (Thermofluor), we found that the mutation does not affect the interaction with CK2ß, the non-catalytic CK2 subunit, and that the thermal stability of the protein is even slightly increased. However, a CK2αLys198Arg crystal structure and its comparison with wild-type structures revealed a significant shift of the anion binding site harboured by the P+1 loop. This observation supports the notion that the Lys198Arg mutation causes an alteration of substrate specificity which we underpinned here with enzymological data.

Molecular Plasticity of Crystalline CK2α' Leads to KN2, a Bivalent Inhibitor of Protein Kinase CK2 with Extraordinary Selectivity.

CK2α and CK2α' are paralogous catalytic subunits of CK2, which belongs to the eukaryotic protein kinases. CK2 promotes tumorigenesis and the spread of pathogenic viruses like SARS-CoV-2 and is thus an attractive drug target. Efforts to develop selective CK2 inhibitors binding offside the ATP site had disclosed the αD pocket in CK2α; its occupation requires large conformational adaptations of the helix αD. As shown here, the αD pocket is accessible also in CK2α', where the necessary structural plasticity can be triggered with suitable ligands even in the crystalline state. A CK2α' structure with an ATP site and an αD pocket ligand guided the design of the bivalent CK2 inhibitor KN2. It binds to CK2 with low nanomolar affinity, is cell-permeable, and suppresses the intracellular phosphorylation of typical CK2 substrates. Kinase profiling revealed a high selectivity of KN2 for CK2 and emphasizes the selectivity-promoting potential of the αD pocket.

De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway.

CSNK2B encodes for casein kinase II subunit beta (CK2ß), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and ß-catenin with mutated CK2ß. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated ß-catenin and consequent absence of active ß-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear ß-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS.

Interaction between CK2α and CK2ß, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface.

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2ß). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2ß as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2ß. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2ß-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2ß interactions is important for the future generation of small molecule drug design.

Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design.

At the first glance CK2alpha, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2alpha the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2alpha structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2alpha structures. In the latter a surprising plasticity of important ATP-binding elements - the interdomain hinge region and the glycine-rich loop - was discovered. In fully active CK2alpha the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the gamma-phospho group from its functional position. In addition to this partially inactive state human CK2alpha was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2beta: if CK2beta blocks this site, the fully active conformation of CK2alpha is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.

Different roles of Enhanced Disease Susceptibility1 (EDS1) bound to and dissociated from Phytoalexin Deficient4 (PAD4) in Arabidopsis immunity.

• Enhanced Disease Susceptibility1 (EDS1) is an important regulator of plant basal and receptor-triggered immunity. Arabidopsis EDS1 interacts with two related proteins, Phytoalexin Deficient4 (PAD4) and Senescence Associated Gene101 (SAG101), whose combined activities are essential for defense signaling. The different sizes and intracellular distributions of EDS1-PAD4 and EDS1-SAG101 complexes in Arabidopsis leaf tissues suggest that they perform nonredundant functions. • The nature and biological relevance of EDS1 interactions with PAD4 and SAG101 were explored using yeast three-hybrid assays, in vitro analysis of recombinant proteins purified from Escherichia coli, and characterization of Arabidopsis transgenic plants expressing an eds1 mutant (eds1(L262P) ) protein which no longer binds PAD4 but retains interaction with SAG101. • EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without additional plant factors. Loss of interaction with EDS1 reduces PAD4 post-transcriptional accumulation, consistent with the EDS1 physical association stabilizing PAD4. The dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection foci. By contrast, an EDS1-PAD4 complex is necessary for basal resistance involving transcriptional up-regulation of PAD4 itself and mobilization of salicylic acid defenses. • Different EDS1 and PAD4 molecular configurations have distinct and separable functions in the plant innate immune response.

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2α and its paralogous isoform CK2α'.

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α'). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open ß4/ß5-loop. Comparisons of these structures with existing CK2α/CK2α' and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α' an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α'-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2ß. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.

Crystallization and preliminary crystallographic analysis of Arabidopsis thaliana EDS1, a key component of plant immunity, in complex with its signalling partner SAG101.

In plants, the nucleocytoplasmic protein EDS1 (Enhanced disease susceptibility1) is an important regulator of innate immunity, coordinating host-cell defence and cell-death programs in response to pathogen attack. Arabidopsis thaliana EDS1 stabilizes and signals together with its partners PAD4 (Phytoalexin deficient4) and SAG101 (Senescence-associated gene101). Characterization of EDS1 molecular configurations in vitro and in vivo points to the formation of structurally and spatially distinct EDS1 homomeric dimers and EDS1 heteromeric complexes with either PAD4 or SAG101 as necessary components of the immune response. EDS1, PAD4 and SAG101 constitute a plant-specific protein family with a unique `EP' (EDS1-PAD4-specific) domain at their C-termini and an N-terminal domain resembling enzymes with an α/ß-hydrolase fold. Here, the expression, purification and crystallization of a functional EDS1 complex formed by EDS1 and SAG101 from Arabidopsis thaliana are reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 101.8, b = 115.9, c = 122.8 Å, and diffracted to 3.5 Šresolution.

Structural basis for the design of bisubstrate inhibitors of protein kinase CK2 provided by complex structures with the substrate-competitive inhibitor heparin.

The Ser/Thr kinase CK2, a member of the superfamily of eukaryotic protein kinases, has an acidophilic substrate profile with the substrate recognition sequence S/T-D/E-X-D/E, and it is inhibited by polyanionic substances like heparin. The latter, a highly sulphated glucosamino glycan composed mainly of repeating 2-O-sulpho-α-l-idopyranuronic acid/N,O6-disulpho-α-d-glucosamine disaccharide units, is the longest known substrate-competitive CK2 inhibitor. The structural basis of CK2's preference for anionic substrates and substrate-competitive inhibitors is only vaguely known which limits the value of the substrate-binding region for the structure-based development of CK2 bisubstrate inhibitors. Here, a tetragonal and a monoclinic co-crystal structure of CK2α, the catalytic subunit of CK2, with a decameric heparin fragment are described. In the tetragonal structure, the heparin molecule binds to the polybasic stretch at the beginning of CK2α's helix αC, whereas in the monoclinic structure it occupies the central substrate-recognition region around the P+1 loop. Together, the structures rationalize the inhibitory efficacy of heparin fragments as a function of chain length. The monoclinic CK2α/heparin structure, in which the heparin fragment is particularly well defined, is the first CK2 structure with an anionic inhibitor of considerable size at the central part of the substrate-recognition site. The bound heparin fragment is so close to the binding site of ATP-competitive inhibitors that it can guide the design of linkers and pave the way to efficient CK2 bisubstrate inhibitors in the future.

Crystallization and preliminary crystallographic analysis of Gre2p, an NADP(+)-dependent alcohol dehydrogenase from Saccharomyces cerevisiae.

Gre2p [Genes de respuesta a estres (stress-response gene)] from Saccharomyces cerevisiae is a monomeric enzyme of 342 amino acids with a molecular weight of 38.1 kDa. The enzyme catalyses both the stereospecific reduction of keto compounds and the oxidation of various hydroxy compounds and alcohols by the simultaneous consumption of the cofactor NADPH and formation of NADP(+). Crystals of a Gre2p complex with NADP(+) were grown using PEG 8000 as a precipitant. They belong to the monoclinic space group P2(1). The current diffraction resolution is 3.2 A. In spite of the monomeric nature of Gre2p in solution, packing and self-rotation calculations revealed the existence of two Gre2p protomers per asymmetric unit related by a twofold noncrystallographic axis.

Structural and Mechanistic Basis of the Inhibitory Potency of Selected 2-Aminothiazole Compounds on Protein Kinase CK2.

Selective inhibitors of protein kinase CK2 with significant cytotoxicity on tumor cells based on a 2-aminothiazole scaffold were described recently. Here, these studies are supplemented with representative CK2α/CK2α' complex structures. They reveal that the 2-aminothiazole-based inhibitors occupy the ATP cavity, whereas preliminary data had indicated an allosteric binding site. The crystal structure findings are corroborated by subsequent enzyme kinetic studies; their atomic-resolution quality provides the basis for future optimization of these promising CK2 inhibitors.