Alessandro Pintar

CX, an algorithm that identifies protruding atoms in proteins

MOTIVATION A simple and fast algorithm is described that calculates a measure of protrusion (cx) for atoms in protein structures, directly useable with the common molecular graphics programs. RESULTS A sphere of predetermined radius is centered around each non-hydrogen atom, and the volume occupied by the protein and the free volume within the sphere (internal and external volumes, respectively) are calculated. Atoms in protruding regions have a high ratio (cx) between the external and the internal volume. The program reads a PDB file, and writes the output in the same format, with cx values in the B factor field. Output structure files can be directly displayed with standard molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB Viewer and colored according to cx values. We show the potential use of this program in the analysis of two protein-protein complexes and in the prediction of limited proteolysis sites in native proteins. AVAILABILITY The algorithm is implemented in a standalone program written in C and its source is freely available at ftp.icgeb.trieste.it/pub/CX or on request from the authors.

Atom Depth as a Descriptor of the Protein Interior

Atom depth, defined as the distance (dpx, A) of a nonhydrogen atom from its closest solvent-accessible protein neighbor, provides a simple but precise description of the protein interior. Mean residue depths can be easily computed and are very sensitive to structural features. From the analysis of the average and maximum atom depths of a set of 136 protein structures, we derive a limit of approximately 200 residues for protein and protein domain size. The average and maximum atom depths in a protein are related to its size but not to the fold type. From the same set of structures, we calculated the mean residue depths for the 20 amino acid types, and show that they correlate well with hydrophobicity scales. We show that dpx values can be used to partition atoms in discrete layers according to their depth and to identify atoms that, although buried, are potential targets for posttranslational modifications like phosphorylation. Finally, we find a correlation between highly conserved residues in structural neighbors of the same fold type, and their mean residue depth in the reference structure.

DPX: for the analysis of the protein core

SUMMARY In order to obtain an accurate description of the protein interior, we describe a simple and fast algorithm that measures the depth of each atom in a protein (dpx), defined as its distance (A) from the closest solvent accessible atom. The program reads a PDB file containing the atomic solvent accessibility in the B-factor field, and writes a file in the same format, where the B-factor field now contains the dpx value. Output structure files can be thus directly displayed with molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB View and colored according to dpx values. AVAILABILITY The algorithm is implemented in a standalone program written in C and its source is freely available at ftp.icgeb.trieste.it/pub/DPX or on request from the authors.

The intracellular region of Notch ligands: does the tail make the difference?

AbstractThe cytoplasmic tail of Notch ligands drives endocytosis, mediates association with proteins implicated in the organization of cell-cell junctions and, through regulated intra-membrane proteolysis, is released from the membrane as a signaling fragment. We survey these findings and discuss the role of Notch ligands intracellular region in bidirectional signaling and possibly in signal modulation in mammals.ReviewersThis article was reviewed by Frank Eisenhaber, L Aravind, and Eugene V. Koonin.

Repeats with variations: Accelerated evolution of the Pin2 family of proteinase inhibitors

The Pin2 genes encode potato type II proteinase inhibitors that act against pathogenic attack. The first examples were found only in the Solanaceae family, but, using new EST and genomic data, we have found 11 homologous genes dispersed through almost the whole range of mono- and di-cotyledonous plants. In contrast to the repetitive precursor sequences of the Solanaceae Pin2 genes, the new homologs have only a single repeat unit. The gene family appears to have evolved from a single-domain ancestral gene through a series of gene-duplication and domain-duplication steps. A number of unequal cross-over and gene conversion events could explain the current gene and domain pattern of the Solanaceae Pin2 subfamily.

Proteins of circularly permuted sequence present within the same organism: The major serine proteinase inhibitor from Capsicum annuum seeds

The major serine proteinase inhibitor from bell pepper (Capsicum annuum, paprika) seeds was isolated, characterized, and sequenced, and its disulfide bond topology was determined. PSI‐1.2 is a 52‐amino‐acid‐long, cysteine‐rich polypeptide that inhibits both trypsin (Ki = 4.6 × 10−9 M) and chymotrypsin (Ki = 1.1 × 10−8 M) and is a circularly permuted member of the potato type II inhibitor family. Mature proteins of this family are produced from precursor proteins containing two to eight repeat units that are proteolytically cleaved within, rather than between, the repeats. In contrast, PSI‐1.2 corresponds to a complete repeat that was predicted as the putative ancestral protein of the potato type II family. To our knowledge, this is the first case in which two proteins related to each other by circular permutation are shown to exist in the same organism and are expressed within the same organ. PSI‐1.2 is not derived from any of the known precursors, and it contains a unique amphiphilic segment in one of its loops. A systematic comparison of the related precursor repeat‐sequences reveals common evolutionary patterns that are in agreement with the ancestral gene‐duplication hypothesis.

CX, DPX and PRIDE: WWW servers for the analysis and comparison of protein 3D structures.

The WWW servers at are dedicated to the analysis of protein 3D structures submitted by the users as the Protein Data Bank (PDB) files. CX computes an atomic protrusion index that makes it possible to highlight the protruding atoms within a protein 3D structure. DPX calculates a depth index for the buried atoms and makes it possible to analyze the distribution of buried residues. CX and DPX return PDB files containing the calculated indices that can then be visualized using standard programs, such as Swiss-PDBviewer and Rasmol. PRIDE compares 3D structures using a fast algorithm based on the distribution of inter-atomic distances. The options include pairwise as well as multiple comparisons, and fold recognition based on searching the CATH fold database.

The intracellular region of the Notch ligand Jagged-1 gains partial structure upon binding to synthetic membranes.

Notch ligands are membrane‐spanning proteins made of a large extracellular region, a transmembrane segment, and a ∼ 100–200 residue cytoplasmic tail. The intracellular region of Jagged‐1, one of the five ligands to Notch receptors in man, mediates protein–protein interactions through the C‐terminal PDZ binding motif, is involved in receptor/ligand endocytosis triggered by mono‐ubiquitination, and, as a consequence of regulated intramembrane proteolysis, can be released into the cytosol as a signaling fragment. The intracellular region of Jagged‐1 may then exist in at least two forms: as a membrane‐tethered protein located at the interface between the membrane and the cytoplasm, and as a soluble nucleocytoplasmic protein. Here, we report the characterization, in different environments, of a recombinant protein corresponding to the human Jagged‐1 intracellular region (J1_tmic). In solution, J1_tmic behaves as an intrinsically disordered protein, but displays a significant helical propensity. In the presence of SDS micelles and phospholipid vesicles, used to mimick the interface between the plasma membrane and the cytosol, J1_tmic undergoes a substantial conformational change. We show that the interaction of J1_tmic with SDS micelles drives partial helix formation, as measured by circular dichroism, and that the helical content depends on pH in a reversible manner. An increase in the helical content is observed also in the presence of vesicles made of negatively charged, but not zwitterionic, phospholipids. We propose that this partial folding may have implications in the interactions of J1_tmic with its binding partners, as well as in its post‐translational modifications.

The interaction of Jagged-1 cytoplasmic tail with afadin PDZ domain is local, folding-independent, and tuned by phosphorylation

Jagged‐1, one of the five Notch ligands in man, is a membrane‐spanning protein made of a large extracellular region and a 125‐residue cytoplasmic tail bearing a C‐terminal PDZ recognition motif (1213RMEYIV1218). Binding of Jagged‐1 intracellular region to the PDZ domain of afadin, a protein located at cell–cell adherens junctions, couples Notch signaling with the adhesion system and the cytoskeleton. Using NMR chemical shift perturbation and surface plasmon resonance, we studied the interaction between the PDZ domain of afadin (AF6_PDZ) and a series of polypeptides comprising the PDZ‐binding motif. Chemical shift mapping of AF6_PDZ upon binding of ligands of different length (6, 24, and 133 residues) showed that the interaction is strictly local and involves only the binding groove in the PDZ. The recombinant protein corresponding to the entire intracellular region of Jagged‐1, J1_ic, is mainly disordered in solution, and chemical shift mapping of J1_ic in the presence of AF6_PDZ showed that binding is not coupled to folding. Binding studies on a series of 24‐residue peptides phosphorylated at different positions showed that phosphorylation of the tyrosine at position ‐2 of the PDZ‐binding motif decreases its affinity for AF6_PDZ, and may play a role in the modulation of this interaction. Finally, we show that the R1213Q mutation located in the PDZ‐binding motif and associated with extrahepatic biliary atresia increases the affinity for AF6_PDZ, suggesting that this syndrome may arise from an imbalance in the coupling of Notch signaling to the cytoskeleton. Copyright

Exon 6 of human Jagged-1 encodes an autonomously folding unit

Human Jagged‐1 is predicted to contain 16 epidermal growth factor‐like (EGF) repeats. The oxidative folding of EGF‐2, despite the several conditions tested, systematically led to complex mixtures. A longer peptide spanning the C‐terminal part of EGF‐1 and the complete EGF‐2 repeat, on the contrary, could be readily refolded. This peptide, which corresponds to the entire exon 6 of the Jagged‐1 gene, thus represents an autonomously folding unit. We show that it is structured in solution, as suggested by circular dichroism and NMR spectroscopy, and displays an EGF‐like disulfide bond topology, as determined by disulfide mapping.