Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2.

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the ß4ß5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the ß regulatory subunits.

Unprecedented selectivity and structural determinants of a new class of protein kinase CK2 inhibitors in clinical trials for the treatment of cancer.

5-(3-Chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer, is representative of a new class of CK2 inhibitors with K(i) values in the low nanomolar range and unprecedented selectivity versus other kinases. Here we present the crystal structure of the complexes of CX-4945 and two analogues (CX-5011 and CX-5279) with the catalytic subunit of human CK2. Consistent with their ATP-competitive mode of inhibition, all three compounds bind in the active site of CK2 (type I inhibitors). The tricyclic scaffold of the inhibitors superposes on the adenine of ATP, establishing multiple hydrophobic interactions with the binding cavity. The more extended scaffold, as compared to that of ATP, allows the carboxylic function, shared by all three ligands, to penetrate into the deepest part of the active site where it makes interactions with conserved water W1 and Lys-68, thus accounting for the crucial role of this negatively charged group in conferring high potency to this class of inhibitors. The presence of a pyrimidine in CX-5011 and in CX-5279 instead of a pyridine (as in CX-4945) ring is likely to account for the higher specificity of these compounds whose Gini coefficients, calculated by profiling them against panels of 102 and/or 235 kinases, are significantly higher than that of CX-4945 (0.735 and 0.755, respectively, vs 0.615), marking the highest selectivity ever reported for CK2 inhibitors.

Structure and immunomodulatory property relationship in NapA of Borrelia burgdorferi.

NapA from Borrelia burgdorferi is a member of the Dps-like protein family with specific immunomodulatory properties; in particular, NapA is able to induce the expression of IL-23 in neutrophils and monocytes, as well as the expression of IL-6, IL-1ß, and transforming growth factor beta (TGF-ß) in monocytes, via Toll-like receptor (TLR) 2. Such an activity on innate immune cells triggers a synovial fluid Th17 response. Here we report the crystal structure of NapA, determined at 2.6Å resolution, which shows that the quaternary structure of the protein is that of a dodecamer with 23 symmetry, typical of the proteins of the family. We also demonstrate that the N- and C-terminal tails, which are flexible and not visible in the crystal, are not relevant for its pro-Th17 activity. Based on the crystal structure and on the comparison with the structure of the orthologous protein from Helicobacter pylori, HP-NAP, we hypothesize that the charge distributions on the two proteins' surfaces are responsible for the interaction with TLR2 and for the different behaviors in modulating the immune response.

Quinalizarin as a potent, selective and cell-permeable inhibitor of protein kinase CK2.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a moderately potent and poorly selective inhibitor of protein kinase CK2, one of the most pleiotropic serine/threonine protein kinases, implicated in neoplasia and in other global diseases. By virtual screening of the MMS (Molecular Modeling Section) database, we have now identified quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) as an inhibitor of CK2 that is more potent and selective than emodin. CK2 inhibition by quinalizarin is competitive with respect to ATP, with a Ki value of approx. 50 nM. Tested at 1 microM concentration on a panel of 75 protein kinases, quinalizarin drastically inhibits only CK2, with a promiscuity score (11.1), which is the lowest ever reported so far for a CK2 inhibitor. Especially remarkable is the ability of quinalizarin to discriminate between CK2 and a number of kinases, notably DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase), PIM (provirus integration site for Moloney murine leukaemia virus) 1, 2 and 3, HIPK2 (homeodomain-interacting protein kinase-2), MNK1 [MAPK (mitogen-activated protein kinase)-interacting kinase 1], ERK8 (extracellular-signal-regulated kinase 8) and PKD1 (protein kinase D 1), which conversely tend to be inhibited as drastically as CK2 by commercially available CK2 inhibitors. The determination of the crystal structure of a complex between quinalizarin and CK2alpha subunit highlights the relevance of polar interactions in stabilizing the binding, an unusual characteristic for a CK2 inhibitor, and disclose other structural features which may account for the narrow selectivity of this compound. Tested on Jurkat cells, quinalizarin proved able to inhibit endogenous CK2 and to induce apoptosis more efficiently than the commonly used CK2 inhibitors TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole).

Borrelia burgdorferi NapA-driven Th17 cell inflammation in lyme arthritis.

OBJECTIVE: Human Lyme arthritis caused by Borrelia burgdorferi is characterized by an inflammatory infiltrate that consists mainly of neutrophils and T cells. This study was undertaken to evaluate the role of the innate and acquired immune responses elicited by the neutrophil-activating protein A (NapA) of B burgdorferi in patients with Lyme arthritis. METHODS: Serum anti-NapA antibodies were measured in 27 patients with Lyme arthritis and 30 healthy control subjects. The cytokine profile of synovial fluid T cells specific for NapA was investigated in 5 patients with Lyme arthritis. The cytokine profile induced by NapA in neutrophils and monocytes was also investigated. RESULTS: Serum anti-NapA antibodies were found in 48% of the patients with Lyme arthritis but were undetectable in the healthy controls. T cells from the synovial fluid of patients with Lyme arthritis produced interleukin-17 (IL-17) in response to NapA. Moreover, NapA was able to induce the expression of IL-23 in neutrophils and monocytes, as well as the expression of IL-6, IL-1beta, and transforming growth factor beta (TGFbeta) in monocytes, via Toll-like receptor 2. CONCLUSION: These findings indicate that NapA of B burgdorferi is able to drive the expression of IL-6, IL-1beta, IL-23, and TGFbeta by cells of the innate immune system and to elicit a synovial fluid Th17 cell response that might play a crucial role in the pathogenesis of Lyme arthritis.

A rapid method for the purification of wild-type and V804M mutant ret catalytic domain: A tool to study thyroid cancer.

RET (rearranged during transfection) is a transmembrane tyrosine kinase and acts as co-receptor of glial-derived neurotrophic factor (GDNF) family neurothrofic factors in complex with GFRalpha family proteins; RET is important for development of enteric nervous system and renal organogenesis during embryonal life. Alterations in Ret gene are related to several neoplasias: point mutations are identified in medullary thyroid carcinoma (MTC) and multiple endocrine neoplasias 2A and B (MEN2A and B), while translocations and chromosomal inversions cause papillary thyroid carcinoma (PTC). We expressed recombinant RET kinase domain (rRET) containing the active site, the ATP binding pocket, and the activation loop with regulatory activity, with the Baculovirus expression system. RET was purified by a two-step procedure consisting of an anion exchange chromatography followed by nickel affinity chromatography. Moreover a biochemical characterization of the recombinant product was performed in order to verify its activity (by ELISA) and physical state (dynamic light scattering). We used rRET to validate an ELISA-based kinase assay, by testing inhibitors reported in literature such as PP1 and PP2. This method represents an easy system to screen potential inhibitors found by computational methods. We also produced V804M mutants to identify inhibitors that can overcome resistance to PP1 and ZD6474. The catalytic domain of RET can be used also for X-ray diffraction to obtain information about the three-dimensional structure, necessary for a rational design of selective inhibitors: it represents an important tool to understand the molecular mechanisms causing thyroid cancer and to care it.

Crystal structure of alkyl hydroperoxide-reductase (AhpC) from Helicobacter pylori.

The AhpC protein from H. pylori, a thioredoxin (Trx)-dependent alkyl hydroperoxide-reductase, is a member of the ubiquitous 2-Cys peroxiredoxins family (2-Cys Prxs), a group of thiol-specific antioxidant enzymes. Prxs exert the protective antioxidant role in cells through their peroxidase activity, whereby hydrogen peroxide, peroxynitrite and a wide range of organic hydroperoxides (ROOH) are reduced and detoxified (ROOH + 2e(-)-->ROH + H2O). In this study AhpC has been cloned and overexpressed in E. coli. After purification to homogeneity, crystals of the recombinant protein were grown. They diffract to 2.95 A resolution using synchrotron radiation. The crystal structure of AhpC has been determined using the molecular replacement method (R = 23.6%, R(free) = 25.9%). The model, similar in the overall to other members of the 2-Cys Prx family crystallized as toroide-shaped complexes, consists of a pentameric arrangement of homodimers [(alpha2)5 decamer]. The model of AhpC from H. pylori presents significant differences with respect to other members of the family: apart from some loop regions, alpha5-helix and the C-terminus is shifted, preventing the C-terminal tail of the second subunit from extending toward this region of the molecule. Oligomerization properties of AhpC have been also characterized by gel filtration chromatography.

Structure of the neutrophil-activating protein from Helicobacter pylori.

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.

The neutrophil-activating protein of Helicobacter pylori.

Infection of the stomach mucosa by the gastric pathogen Helicobacter pylori is accompanied by a large infiltration of neutrophils and monocytes which are believed to contribute substantially to H. pylori-induced gastritis. A protein was identified (HP-NAP for neutrophil-activating protein from H. pylori) that was capable of increasing the adhesion of neutrophils to endothelial cells. We have demonstrated that HP-NAP is a dodecamer composed of identical 17-kDa subunits that induces the production of reactive oxygen radicals (ROIs) by neutrophils via a cascade of intracellular activation events. HP-NAP has also been shown to be chemotactic for neutrophils and monocytes, and a majority of H. pylori-infected patients have been found to produce antibodies specific for HP-NAP making it a strong vaccine candidate. More recently it has been shown that HP-NAP can stimulate tissue factor and plasminogen activator inhibitor-2 production by human monocytes. While structurally similar to the Escherichia coli DNA-binding protein Dps, HP-NAP has characteristics that are more similar to bacterioferritins being capable of binding up to 500 atoms of iron in vitro. Further study, however, has revealed that synthesis of HP-NAP in H. pylori is not altered by the addition or subtraction of metal ions from its growth medium suggesting that the primary role of the protein in vivo is not as a metal-binding protein. A number of other reports have proposed that HP-NAP acts as an adhesin being capable of binding several different compounds in vitro. Sequence analysis of the genomes of several other bacteria reveal that many possess Dps/HP-NAP-like proteins. The preliminary characterisation of some of these proteins will be discussed.

Structure of two iron-binding proteins from Bacillus anthracis.

Bacillus anthracis is currently under intense investigation due to its primary importance as a human pathogen. Particularly important is the development of novel anti-anthrax vaccines, devoid of the current side effects. A novel class of immunogenic bacterial proteins consists of dodecamers homologous to the DNA-binding protein of Escherichia coli (Dps). Two Dps homologous genes are present in the B. anthracis genome. The crystal structures of these two proteins (Dlp-1 and Dlp-2) have been determined and are presented here. They are sphere-like proteins with an internal cavity. We also show that they act as ferritins and are thus involved in iron uptake and regulation, a fundamental function during bacterial growth.