Structural biology

Field of Interest

The scientific activity of the laboratory is focused on the crystal structure determination of proteins, often in collaboration with other research groups of VIMM. In the overall since January 2005, 43 (plus 5 on hold) structural data sets have been deposited at the Protein Data Bank (www.rcsb.org).

Summary of research activity (2005-2009)

• Several proteins of the Helicobacter pylori pathogen have been cloned and their crystal structure determined. In particular, some members of the cag pathogenicity island (CagS and CagD), alkyl hydroperoxide-reductase (AhpC), superoxide dismutase, YbgC thioesterase, TenA, YceI

Figure 1. Space filling model of the dimer of copper amine oxidase from bovine serum. Protein atoms of the two subunits are in green and cyan, sugar residues in yellow.
Figure 1. Space filling model of the dimer of copper amine oxidase from bovine serum. Protein atoms of the two subunits are in green and cyan, sugar residues in yellow.
[click image to enlarge]
• The 3D structure of various enzymes involved in the purine metabolism has been determined: 5-hydroxyisourate hydrolase, OHCU decarboxylase, Puue allantoinase, S-ureidoglycine:glyoxalate aminotransferase, either alone or in complex with inhibitors.
• The SERCA pump from bovine muscle has been crystallized and its structure determined.
• A combined structural and computational approach has allowed the definition of the characteristics of the amyloidogenicity of transthyretin mutants.
• Other protein structures determined:
  • amine oxidase from bovine serum and amine oxidase/inhibitor complexes;
  • TpF1 from Treponema pallidum and NAPA from Borrelia burgdorferi;
  • allergenic protein Pru p 3 from peach;
  • bovine 3-hydroxyanthranilate 3,4-dioxygenase;
  • inhibitor/CK2 complexes.

On-going activities and future research plans

The scientific activity of the group is focused on three main projects, regarding Helicobacter pylori proteins, SERCA Ca²⁺ pump and the structure of enzymes involved in the uric acid degradation pathway. In addition, there are active collaborations with the groups of Prof. Lorenzo Pinna and Roberto Battistutta on protein kinases inhibitors, and with Prof. Rodolfo Berni (Univ. of Parma) on amyloidogenic mutants of TTR.

Structural proteomics of Helicobacter pylori

H. pylori chronically infects the gastric mucosa of the majority of the human population, and it is implicated in the development of severe gastroduodenal diseases, including active chronic gastritis, peptic ulcers, gastric adenocarcinoma and mucosa-associated lymphoma. This bacterium is interesting not only because it is a human pathogen not totally understood, but also for other reasons: it lives only in a very special niche, the human stomach, and during evolution he has adapted to this peculiar and hard environment. Its genome (the genomes of three different strains have been sequenced) codes for about 1500 hypothetical genes, but bioinformatics analysis allows to assign a tentative function to about only 2/3 of these genes, suggesting a specialized and peculiar evolution of HP with respect to other well known gram-negative bacteria, like E. coli. We plan to use HP as a paradigm of a special, relatively simple prokaryote and to try to define all its components in terms of molecular structures, functions and inter-relationships. In the past, we have determined the crystal structure of the neutrophil activating protein (NAP, in collaboration with Cesare Montecucco's group), a 200 kDa oligomer capable of promoting neutrophil adhesion to endothelial cells and of inducing neutrophils to produce reactive oxygen radicals, and of alkyl hydroperoxide-reductase.

The cag pathogenicity island. Type I strains of the bacterium contain a 37 kb foreign DNA region, called cag pathogenicity island, involved in virulence (cag PAI). The cag-PAI encodes a functional type IV secretion apparatus homologous to the VirB/D4 type IV secretion system (T4SS) of the plant pathogen Agrobacterium tumefaciens. The type IV secretion systems of Gram-negative bacteria are involved in the translocation of DNA and/or effector proteins to infected eukaryotic host cells. The T4SS encoded by the cag-PAI of H. pylori is responsible for the translocation into the host cells of the protein CagA, a major antigenic virulence factor of this bacterium. Little is known about the function of the other proteins encoded by the cag-PAI. In the last years we have determined various structures (all were determined using the Se-Met technique), including CagZ, a 23 kDa protein composed of seven α-helices, CagS, an all-α structure, and CagD, a covalent dimer in which each monomer folds as a single domain, composed of five β-strands and three α-helices. We show that CagD is a crucial component of the T4SS that is involved in CagA translocation into the host epithelial cells; however, it does not seem absolutely necessary for pilus assembly. Several others Cag proteins have been cloned and expressed and crystallization tests are under way.

Figure 2. Ribbon drawing of the dimer of YceI (HP1286) with erucamide bound inside the cavity.

Figure 2. Ribbon drawing of the dimer of YceI (HP1286) with erucamide bound inside the cavity.
[click image to enlarge]

H. pylori proteins involved in colonization and persistence. Recently, new genes involved in H. pylori stomach colonization were identified by mutagenesis (Baldwin et al. 2007). Of the loci measured, 223 (29%) had a predicted colonization effect. These included previously described H. pylori virulence genes, genes implicated in virulence in other pathogenic bacteria, and 81 hypothetical proteins. A subset of these genes were cloned and some of the coded proteins crystallized. We have determined the crystal structures of alkyl-hydroperoxide-reductase (AhpC, HP1563), YbcG (HP0496, in collaboration with L. Terradot), superoxide dismutase (SodB, HP0389, in collaboration with A. Zagari group), TenA (HP1287), HP-YceI (HP1286). Others have been cloned and their structure is on the way of being solved.

Structural characterization of Ca²⁺-pumps
(in collaboration with Prof. Ernesto Carafoli's group)

Figure 3. Ribbon representation of SERCA from bovine muscle, E1 form. Ions bound are shown as green spheres, AMP-PNP in red.

Figure 3. Ribbon representation of SERCA from bovine muscle, E1 form. Ions bound are shown as green spheres, AMP-PNP in red.
[click image to enlarge]

In mammals, three Ca-ATPases have been described: the sarcoplasmic reticulum (SERCA), plasma membrane (PMCA) and the Golgi network (SPCA) pumps (Brini and Carafoli, 2009). The first, SERCA pump, controls the concentration of cytoplasmic Ca²⁺ in muscle cells, where its ability to remove Ca²⁺ from the cytosol induces relaxation. Two human diseases have been associated with SERCA pump gene defects, Brody's and Darier's disease. The former is a rare recessive muscular condition characterized by impaired relaxation, painless cramps and stiffness. At least three mutations have been identified as responsible for the pathology. Darier's disease is a rare autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells and abnormal keratinization. Finally, SERCA is functionally decreased in nearly all models of heart failure and mutations of the SERCA2a protein may predispose to it. The crystal structure of the SERCA pump from rabbit skeletal muscle in different states is known (Toyoshima et al., 2000, 2002, 2004; Lykke-Moller Sorensen et al., 2004). On the contrary, no crystal structure of its pathological mutants is available. A major reason is that the protein crystallized was extracted from rabbit muscle. In order to study the effects of point mutations on the protein conformation and behavior and to understand the molecular aspects of the pathologies, we are undertaking a structural and functional study of the bovine and human SERCA proteins and of their mutants.
Recently, cattle belonging to the Chianina family with a SERCA pump mutated at residue R164 have been identified. This mutation induces in the animal symptoms very similar to those of the Brody syndrome, suggesting that these animals can be used as animal models of the human disease. Using a procedure similar to that used for rabbit muscle, crystals of bovine wild-type SERCA have been obtained in our lab. The molecular model is shown in the figure. Crystallization trials of the R164H mutant protein are in progress and other mutants are planned.

Enzymes involved in purine metabolism
(in collaboration with R. Berni and R. Percudani, Univ. of Parma)

Very recently, the complete metabolic degradation of uric acid has been described: two enzymes, that catalyze the conversion of 5-hydroxyisourate (HIU) to allantoin, have been identified. The discovery of these two enzymes complete the degradation pathway of urate, in addition to the already known urate oxidase. The two new enzymes were HIU hydrolase (HIUase) and OHCU decarboxylase. The first enzyme of this pathway was already structurally and functionally characterized, whereas the crystal structure of the other two, HIUase and OHCU decarboxylase, has been determined by our group. In addition, the crystal structure of S-ureidoglycine:glyoxylate aminotransferase and of a PuuE allantoinase analog have been solved.

Other proteins

Structural studies are in progress on other protein systems relevant for human health. In particular, Site 1 protease (S1P), a protease implicated in the processing of the glycoproteins of Old World arenaviruses and South American hemorrhagic fever viruses (In collaboration with S. Kunz, Lausanne); and Sesame 2S albumins (Ses i 1 and 2), small proteins responsible of food allergy (In collaboration with V. De Filippis, and V. Castagliuolo, Padua).

All these studies make use of the facilities at VIMM along with those available at the Dept of Chemistry of the University of Padua. Diffraction data will be measured at the X-ray diffraction beam-lines of the Synchrotrons ESRF (Grenoble) and Elettra (Trieste).

Synoptic CV

Appointments

Honours

** Selected Publications (VIMM)

** Other Publications (VIMM)

** Additional Publications

Selected Seminars

Contact

Giuseppe Zanotti Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Last updated: 28/03/2010, GZ ·