Structural basis of molecular mechanisms and functions: oncogenic protein kinases and SLC26 anion transporters

Field of Interest

The research of this group concerns with the study of the architecture and structure of proteins, to elucidate the basic mechanisms underlying the biological function at molecular level. The main experimental technique that we use is macromolecular crystallography, along with classical biophysical methods such as circular dichroism, fluorescence, dynamic light scattering (DLS) and calorimetry.

On-going activities and future research plans

The two main projects of this unit are the following.

Structural basis of the inhibition of pathogenic protein kinases

The enzymes catalyzing protein phosphorylation in cells, termed protein kinases (PKs), share a conserved catalytic domain and play a paramount role in signal transduction. Deregulation of PKs often underlies pathologies making these enzymes attractive targets for drugs to treat cancer and other global diseases, notably diabetes, inflammatory and infectious diseases, stroke, hypertension and Alzheimer's.

Figure 1. Protein kinase CK2 in complex with the inhibitor tyrphostin. The protein surface is coloured based on the electrostatics (blue positive charges, red negative charges). Tyrphostin (shown in sticks, carbon atoms in green), as the other selective inhibitors, binds into the deep ATP-binding cleft.

Figure 1. Protein kinase CK2 in complex with the inhibitor tyrphostin. The protein surface is coloured based on the electrostatics (blue positive charges, red negative charges). Tyrphostin (shown in sticks, carbon atoms in green), as the other selective inhibitors, binds into the deep ATP-binding cleft.
[click image to enlarge]

The goal of this project is the structural characterization of the protein function and of the inhibition mechanism of powerful and selective compounds for potentially pathogenic protein kinases. The derived information is the basis for the 3D structure-guided rational design, virtual screening and high throughput docking aimed at the discovery of new inhibitors (in collaboration with Prof. Stefano Moro, University of Padua). The biochemical characterization of inhibition constants and selectivity by means of in vitro and in cell assays are performed in collaboration with Prof. Flavio Meggio (University of Padua) and Prof. Lorenzo A. Pinna (VIMM). Currently the protein kinase under study is CK2. CK2 unusually high constitutive activity is suspected to enhance the tumour phenotype by supporting cell survival "at all costs". In the last years our group has determined the crystal structures of CK2 in complex with several inhibitors belonging to different families of compounds. This has allowed to elucidate some relevant features of these inhibitors and of the CK2 active site and to explain why they are active and selective against CK2. The inhibitors studied are derivatives of benzo(triazoles/imidazoles), anthraquinones, quinazolinones and coumarins, with inhibitory constants or IC50 in the low micromolar or nanomolar range.

Perspective

Crystallization and structure determination of functionally relevant forms of CK2 and of complexes with optimized inhibitors, carrying both known and new scaffolds.

Relevance

We are accumulating a large amount of information on the molecular aspects of the protein function and on the enzyme-inhibitors interaction. The latter can be used to design new molecules in order to increase the potency and the specificity of binding, along as the drug-like properties of these molecules.

Structural characterization of SulP anion transporters

The long-term aim of this project is the structural and functional characterization of members of the SulP family of anion transporters, among which SLC26A5, prestin. The solute linked carrier 26, SLC26, is a gene family of versatile anion exchangers with intriguing roles in normal physiology and human pathophysiology. Prestin is expressed in the inner ear and mutations are linked to hearing impairment. To reach this final goal we intend to use the tools of macromolecular X-ray crystallography, an appropriate technique to gain the detailed structural information needed. Due to the low abundance of the SLC26 transporters from natural sources, the unavoidable choice is the production of recombinant material.

Perspective

We want to derive a model for the structural organization of SulP transporters which will provide insights into the functional consequences of mutations linked to genetic diseases. This model will also be used as a template for the members of the SLC26 gene family of anion transporters.

Relevance

This family of anion transporters has not been structurally characterized yet and the mechanism of action is still substantially unknown. The clinical relevance of this gene family has been highlighted with the identification of pathogenetic mutations in four of its genes, namely SLC26A2, A3, A4 and A5, that have been shown to lead to rare human disorders of proven genetic origin. In particular, SLC26A5 mutations are identified in individuals with recessive non-syndromic deafness. Most of the knowledge related to the SLC26 members has come from studies within the last decade and the increasing interest in these transporters is testified by the growing number of publications within recent years.

Synoptic CV

Selected VIMM Publications

• Battistutta R (2009) Protein kinase CK2 in health and disease: Structural bases of protein kinase CK2 inhibition. Cell. Mol. Life Sci. 66:1868-89.
• Mazzorana M, Pinna LA, Battistutta R (2008) A structural insight into CK2 inhibition. Mol. Cell. Biochem. 316:57-62.
• Pasqualetto E, Seydel A, Pellini A, Battistutta R (2008) Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin. Protein Expr. Purif. 58:249-56.
• Battistutta R, Mazzorana M, Cendron L, Bortolato A, Sarno S, Kazimierczuk Z, Zanotti G, Moro S, Pinna LA (2007) The ATP-binding site of protein kinase CK2 holds a positive electrostatic area and conserved water molecules. Chembiochem 8:1804-9.
• Battistutta R, Mazzorana M, Sarno S, Kazimierczuk Z, Zanotti G, Pinna LA (2005) Inspecting the structure-activity relationship of protein kinase CK2 inhibitors derived from tetrabromo-benzimidazole. Chem. Biol. 12:1211-9.

VIMM Publications

• Cozza G, Mazzorana M, Papinutto E, Bain J, Elliott M, di Maira G, Gianoncelli A, Pagano MA, Sarno S, Ruzzene M, Battistutta R, Meggio F, Moro S, Zagotto G, Pinna LA (2009) Quinalizarin as a potent, selective and cell-permeable inhibitor of protein kinase CK2. Biochem. J. 421:387-95.
• Battistutta R (2009) Protein kinase CK2 in health and disease: Structural bases of protein kinase CK2 inhibition. Cell. Mol. Life Sci. 66:1868-89.
• Mazzorana M, Pinna LA, Battistutta R (2008) A structural insight into CK2 inhibition. Mol. Cell. Biochem. 316:57-62.
• Chilin A, Battistutta R, Bortolato A, Cozza G, Zanatta S, Poletto G, Mazzorana M, Zagotto G, Uriarte E, Guiotto A, Pinna LA, Meggio F, Moro S (2008) Coumarin as attractive casein kinase 2 (CK2) inhibitor scaffold: an integrate approach to elucidate the putative binding motif and explain structure-activity relationships. J. Med. Chem. 51:752-9.
• Pasqualetto E, Seydel A, Pellini A, Battistutta R (2008) Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin. Protein Expr. Purif. 58:249-56.
• Battistutta R, Mazzorana M, Cendron L, Bortolato A, Sarno S, Kazimierczuk Z, Zanotti G, Moro S, Pinna LA (2007) The ATP-binding site of protein kinase CK2 holds a positive electrostatic area and conserved water molecules. Chembiochem 8:1804-9.
• Cendron L, Tasca E, Seraglio T, Seydel A, Angelini A, Battistutta R, Montecucco C, Zanotti G (2007) The crystal structure of CagS from the Helicobacter pylori pathogenicity island. Proteins 69:440-3.
• Tosatto SC, Battistutta R (2007) TAP score: torsion angle propensity normalization applied to local protein structure evaluation. BMC Bioinformatics 8:155.
• Genisset C, Galeotti CL, Lupetti P, Mercati D, Skibinski DA, Barone S, Battistutta R, de Bernard M, Telford JL (2006) A Helicobacter pylori vacuolating toxin mutant that fails to oligomerize has a dominant negative phenotype. Infect. Immun. 74:1786-94.
• Thumiger A, Polenghi A, Papinutto E, Battistutta R, Montecucco C, Zanotti G (2006) Crystal structure of antigen TpF1 from Treponema pallidum. Proteins 62:827-30.
• Battistutta R, Mazzorana M, Sarno S, Kazimierczuk Z, Zanotti G, Pinna LA (2005) Inspecting the structure-activity relationship of protein kinase CK2 inhibitors derived from tetrabromo-benzimidazole. Chem. Biol. 12:1211-9.
• Battistutta R, Mazzorana M, Sarno S, Kazimierczuk Z, Zanotti G, Pinna LA (2005) Inspecting the structure-activity relationship of protein kinase CK2 inhibitors derived from tetrabromo-benzimidazole. Chem. Biol. 12:1211-9.
• Papinutto E, Windle HJ, Cendron L, Battistutta R, Kelleher D, Zanotti G (2005) Crystal structure of alkyl hydroperoxide-reductase (AhpC) from Helicobacter pylori. Biochim. Biophys. Acta 1753:240-6.
• Sarno S, Salvi M, Battistutta R, Zanotti G, Pinna LA (2005) Features and potentials of ATP-site directed CK2 inhibitors. Biochim. Biophys. Acta 1754:263-70.
• Lunelli M, Di Paolo ML, Biadene M, Calderone V, Battistutta R, Scarpa M, Rigo A, Zanotti G (2005) Crystal structure of amine oxidase from bovine serum. J. Mol. Biol. 346:991-1004.
• Schievano E, Calisti T, Menegazzo I, Battistutta R, Peggion E, Mammi S, Palù G, Loregian A (2004) pH-Dependent conformational changes and topology of a herpesvirus translocating peptide in a membrane-mimetic environment. Biochemistry 43:9343-51.
• Cendron L, Seydel A, Angelini A, Battistutta R, Zanotti G (2004) Crystal structure of CagZ, a protein from the Helicobacter pylori pathogenicity island that encodes for a type IV secretion system. J. Mol. Biol. 340:881-9.

Selected Seminars

Contact

Roberto Battistutta Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 236 Fax:  (+39) 049 7923 250

Last updated: 28/03/2010, RB ·