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C Savino

Publications and source records attributed to C Savino.

13 recordsLinked to original sources
PubMed2003

The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense.

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that protect plants from fungal invasion. They interact with endopolygalacturonases secreted by phytopathogenic fungi, inhibit their enzymatic activity, and favor the accumulation of oligogalacturonides, which activate plant defense responses. PGIPs are members of the leucine-rich repeat (LRR) protein family that in plants play crucial roles in development, defense against pathogens, and recognition of beneficial microbes. Here we report the crystal structure at 1.7-A resolution of a PGIP from Phaseolus vulgaris. The structure is characterized by the presence of two beta-sheets instead of the single one originally predicted by modeling studies. The structure also reveals a negatively charged surface on the LRR concave face, likely involved in binding polygalacturonases. The structural information on PGIP provides a basis for designing more efficient inhibitors for plant protection.

Amino Acid Sequence↗
PubMed2001

Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein).

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 A resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding.

Base Sequence↗
PubMed2000

The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome.

The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.

Amino Acid Motifs↗
PubMed2000

Engineering His(E7) affects the control of heme reactivity in Aplysia limacina myoglobin.

Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 A crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.

Animals↗
PubMed2000

Crystallization and preliminary X-ray diffraction studies of a monooxygenase from Streptomyces coelicolor A3(2) involved in the biosynthesis of the polyketide actinorhodin.

The aromatic monooxygenase ActVA-Orf6 from Streptomyces coelicolor A3(2) that catalyses an unusual oxidation on the actinorhodin biosynthetic pathway has been crystallized. The crystals diffract to 1.73 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.95, b = 59.29, c = 71.67 A. Solvent-content (44%) and self-rotation function calculations predict the presence of two molecules in the asymmetric unit. Structure determination should provide further insight into the enzyme mechanism and aid in the design of biosynthetic pathways to produce new polyketide natural products with novel functionality.

Anthraquinones↗
PubMed1999

Crystal structure of Trematomus newnesi haemoglobin re-opens the root effect question.

As new structural data have become available, somewhat contrasting explanations of the Root effect in fish haemoglobins (Hb) have been provided. Hb 1 of the Antarctic fish Trematomus newnesi has a nearly pH-independent oxygen affinity, in spite of 95 % sequence identity with Hb 1 of Trematomus (previously named Pagothenia) bernacchii that has a strong Root effect. Here, the 2.2 A R-state structure of Trematomus newnesi Hb 1 is presented. The structure is similar to that of Root effect fish Hbs from Spot and T. bernacchii, suggesting that the differences in the pH dependence cannot be related to the modulation of the R-state. In comparison to T. bernacchii Hb 1, the role of the three mutations Thr41 (C6)alpha-->Ile, Ala97 (G3)alpha-->Ser and His41 (C7)beta-->Tyr at the alpha1beta2-interface is discussed.

Animals↗
PubMed1999

Structural dynamics of ligand diffusion in the protein matrix: A study on a new myoglobin mutant Y(B10) Q(E7) R(E10).

A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O2, and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O2 delivery to tissue at a rate compatible with physiology.

Animals↗
PubMed1999

Crystallization and preliminary X-ray crystallographic analysis of the unusual ferritin from Listeria innocua.

Single crystals of ferritin extracted from Listeria innocua have been obtained by the vapour-diffusion method using PEG 1000 as precipitant. The crystals are orthorhombic, space group P212121, with unit-cell dimensions a = 87.7, b = 137.5, c = 173.1 A. The crystals diffract to 2.9 A resolution on a rotating-anode X-ray source and to 2.35 A resolution on a synchrotron X-ray source. The asymmetric unit contains one molecule formed by 12 subunits, corresponding to a packing density of 2.41 A3 Da-1

Crystallization↗
PubMed1999

Crystallization and preliminary X-ray diffraction study of the endo-polygalacturonase from Fusarium moniliforme.

Endo-polygalacturonases catalyze the fragmentation and solubilization of the homogalacturonan of the plant cell wall. These enzymes are extracellularly targeted glycoproteins produced by a number of organisms such as fungi, bacteria and plants, and are involved in both pathological and physiological processes. Single crystals of the endo-polygalacturonase from the phytopathogenic fungus Fusarium moniliforme were obtained by the vapour-diffusion method at 294 K. The starting material as well as the crystal consist of three forms with different degrees of glycosylation. The crystals belong to the orthorhombic space group P212121 and diffract to 1.9 A resolution on a synchrotron-radiation source under cryocooling conditions.

Crystallization↗
PubMed1998

Crystallization and preliminary X-ray diffraction studies of DNA polymerase from the thermophilic archaeon Sulfolobus solfataricus.

The thermophilic and thermostable family B DNA polymerase from the archaeon Sulfolobus solfataricus (Mr of about 100 kDa) has been crystallized by the hanging-drop vapour-diffusion method at 294 K using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution on a rotating-anode X-ray source. Native data have been collected at 100 K. A heavy-atom derivative search is in progress.

Bacterial Proteins↗
PubMed1998

Crystallization and preliminary X-ray study of saporin, a ribosome-inactivating protein from Saponaria officinalis.

Single crystals of the protein saporin isolated from the seeds of S. officinalis have been grown by the vapor-diffusion method using ammonium sulfate as precipitant. The crystals are tetragonal, space group P4122 (P4322), with cell dimensions a = b = 67.53 and c = 119. 67 A, and diffract to 2.0 A resolution on a rotating-anode X-ray source. The asymmetric unit contains one molecule, corresponding to a volume of the asymmetric unit per unit mass (Vm) of 2.38 A3 Da-1.

Crystallization↗
PubMed1998

Integrating clinical and laboratory data in genetic studies of complex phenotypes: a network-based data management system.

The identification of genes underlying a complex phenotype can be a massive undertaking, and may require a much larger sample size than thought previously. The integration of such large volumes of clinical and laboratory data has become a major challenge. In this paper we describe a network-based data management system designed to address this challenge. Our system offers several advantages. Since the system uses commercial software, it obviates the acquisition, installation, and debugging of privately-available software, and is fully compatible with Windows and other commercial software. The system uses relational database architecture, which offers exceptional flexibility, facilitates complex data queries, and expedites extensive data quality control. The system is particularly designed to integrate clinical and laboratory data efficiently, producing summary reports, pedigrees, and exported files containing both phenotype and genotype data in a virtually unlimited range of formats. We describe a comprehensive system that manages clinical, DNA, cell line, and genotype data, but since the system is modular, researchers can set up only those elements which they need immediately, expanding later as needed.

Clinical Laboratory Information Systems↗