A structural genomics approach to sars coronavirus infection

The S1 and S2 subunits of the spike glycoprotein of SARS-CoV coronavirus have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the similarity, generally exhibited by coronaviruses, of their genomic organisation. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. In facts, all the possible cystine bridges, ten and four disulphide bridges respectively in the S1 and S2 subunits, can be formed in the two proposed models, since always the corresponding cysteine C atoms are found at a maximum distance of 10 Å. Furthermore, only three out of the total fourteen predicted N glycosilation sites of S1 exhibit low surface accessibility and all of the five possible N glycosilation sites of S2 result to be exposed. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity and the overall assembly of the SARS coronavirus peplomer has been performed. A trimer of S1-S2 heterodimers has been modelled on the basis of geometrical consideration, as well as charge and hydrophobicity complementarities. The modelled peplomer accounts for all the functional data so far available for this critical part of the viru

A structural genomics approach to sars coronavirus infection

The S1 and S2 subunits of the spike glycoprotein of SARS-CoV coronavirus have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the similarity, generally exhibited by coronaviruses, of their genomic organisation. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. In facts, all the possible cystine bridges, ten and four disulphide bridges respectively in the S1 and S2 subunits, can be formed in the two proposed models, since always the corresponding cysteine C atoms are found at a maximum distance of 10 Å. Furthermore, only three out of the total fourteen predicted N glycosilation sites of S1 exhibit low surface accessibility and all of the five possible N glycosilation sites of S2 result to be exposed. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity and the overall assembly of the SARS coronavirus peplomer has been performed. A trimer of S1-S2 heterodimers has been modelled on the basis of geometrical consideration, as well as charge and hydrophobicity complementarities. The modelled peplomer accounts for all the functional data so far available for this critical part of the viru

Three-dimensional Computation of Atom Depth in Complex Molecular Structures

Motivation: For a complex molecular system the delineation of atom-atom contacts, exposed surface and binding sites represents a fundamental step to predict its interaction with solvent, ligands and other molecules. Recently, atom depth has been also considered as an additional structural descriptor to correlate protein structure with folding and functional properties. The distance between an atom and the nearest water molecule or the closest surface dot has been proposed as a measure of the atom depth, but, in both cases, the three-dimensional character of depth is largely lost. To calculate atom depths in a way that the molecular shape can be taken into account, a new approach is here proposed. Results: An algorithm has been developed to calculate intersections between the molecular volume and spheres centered on the atoms whose depth has to be quantified. Many proteins with different size and shape have been chosen to compare the results obtained from distance-based and volume– based depth calculations. From the wealth of experimental data available for hen egg white lysozyme, H/D exchange rates and TEMPOL induced paramagnetic perturbations have been analyzed both in terms of depth indexes and of atom distances to the solvent accessible surface. The algorithm here proposed yields better correlations between experimental data and atom depth, particularly for those atoms which are located near to the protein surface. Availability: Instructions to obtain source code and the executable program are available eithe

Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein

The S1 and S2 subunits of the spike glycoprotein of the coronavirus which is responsible for the severe acute respiratory syndrome (SARS) have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures. An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the genomic organisation similarity generally exhibited by coronaviruses. The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein. The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity. © 2003 Elsevier Inc. All rights reserved

Surface accessibility of hew lysozyme

INTRODUCTION: Nowadays structural biology represents an advanced frontier for a detailed understanding of life processes. Several projects of structural genomics are presented world-wide, since predictable or experimentally derived structures, mostly of proteins, represent a solid basis for engineering new therapeutic or diagnostic tools. However, some missing details still exist as the biological functions result to be not simply correlated to protein shapes and flexibility. The presence of “hot spots” on the molecular surface, always including the protein active sites (1,2), and the anomalous activity of some non-competitive enzyme inhibitors (3) suggest that around the protein surface a non-uniform motion of solvent and other molecules occurs. NMR seems to be perfectly suited to investigate on this new dimension of structural biology, since two techniques, i.e. water-protein Overhauser (4) effects and paramagnetic perturbation profiles (5), independently or in a combined way can be used to probe the accessibility of the protein surface towards molecules with different polarity. The results obtained from both NMR methods for a series of small proteins such as BPTI, tendamistat and single chain monellin (5,6) suggest that only a limited number of water molecules reside at the protein surface for a time long enough to yield sizeable intermolecular NOEs. The active sites of these proteins never have such tightly bound water molecules nearby. Accordingly, TEMPOL, a stable uncharged nitroxide, efficiently approaches the latter protein moieties due to the reduced hindrance from solvent molecules. Furthermore, there are protein regions where bound water molecules, organised in strong hydration sites, prevent the access of the nitroxide. MATERIALS AND METHODS: 13C-1H HSQC, 1D and 2D ePHOGSY spectra of 5 mM HEW lysozyme were acquired at 308 K and pH 3.8, using a Bruker Avance 600 spectrometer equipped with a Silicon Graphics Indy workstation. Paramagnetic attenuations were induced by the presence of 50 mM TEMPOL (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl). The enzyme inhibitor (NAG)3 was 7.5 mM to ensure full complexation of lysozyme. RESULTS: Paramagnetic attenuation profiles and water-protein nuclear Overhaser effects observed for HEW lysozyme, obtained in the presence and in the absence of the competitive inhibitor (NAG)3, were analysed in terms of accessibility of water and TEMPOL molecules towards the protein surface. The combined use of ePHOGSY spectroscopy and TEMPOL induced perturbations resulted to be very powerful also to find, as in the previously reported case for BPTI (5), the internal water molecules

NMR studies on the surface accessibility of the archaeal protein Sso7d by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes

Understanding how proteins are approached by surrounding molecules is fundamental to increase our knowledge of life at atomic resolution. Here, the surface accessibility of a multifunctional small protein, the archaeal protein Sso7d from Sulfolobus solfataricus, has been investigated by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes. The DNA binding domain of Sso7d appears very accessible both to TEMPOL and Gd(III)(DTPA-BMA). Differences in paramagnetic attenuation profiles of 1H-15N HSQC protein backbone amide correlations, observed in the presence of the latter paramagnetic probes, are consistent with the hydrogen bond acceptor capability of the N-oxyl moiety of TEMPOL to surface exposed Sso7d amide groups. By using the gadolinium complex as a paramagnetic probe a better agreement between Sso7d structural features and attenuation profile is achieved. It is interesting to note that the protein P-loop region, in spite of the high surface exposure predicted by the available protein structures, is not approached by TEMPOL and only partially by Gd(III)(DTPA-BMA). © 2008 Elsevier B.V. All rights reserved

NMR studies of BPTI aggregation by using paramagnetic relaxation reagents

Paramagnetic probes, whose approach to proteins can be monitored by nuclear magnetic resonance (NMR) studies, have been found of primary relevance for investigating protein surfaces accessibility. Here, paramagnetic probes are also suggested for a systematic investigation on protein aggregation. Bovin pancreatic trypsin inhibitor (BPTI) was used as a model system for aggregation by analyzing its interaction with TEMPOL and Gd(III)DTPA-BMA. Some of the measured paramagnetic relaxation rates of BPTI protons exhibited a reverse dependence on protein concentration, which can be attributed to the formation of transient BPTI aggregates. © 2006 Elsevier B.V. All rights reserved

Structurally Driven Selection of Human Hepatitis C Virus Mimotopes

A structural genomics approach is proposed for the development of new diagnostic kits. It combines molecular modelling, peptide synthesis and immunological tests. The preliminary step is the development of a reliable three-dimensional structure of an immunodominant protein of the target pathogenic organism using the various bioinformatic strategies that are now available to structural biologists. Once the protein structure is obtained, the most surface-exposed fragments with minimal sequence variability among the different strains reported in the genomic data bank are reproduced synthetically as linear peptides. These peptides are then tested for immunoreactivity with the plasma of infected patients to determine whether the synthetic molecules have antigenic activity and can therefore be used to detect infecting agents. This structurally driven selection of mimotopes was successfully performed for the human hepatitis C virus, as five peptides that specifically interact with the plasma of HCV-infected patients were identified solely on the basis of the three-dimensional structure predicted for the E2 homodimer of the 1a viral subtype. A similar approach could easily be extended to a large variety of immunogenic proteins from other pathogenic organisms. </jats:p