The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA.

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.

Flexibility of the PDZ-binding motif in the micelle-bound form of Jagged-1 cytoplasmic tail.

Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a large extracellular region and a 125-residue cytoplasmic tail which bears a C-terminal PDZ recognition motif. To investigate the interaction between Jagged-1 cytoplasmic tail and the inner leaflet of the plasma membrane we determined, by solution NMR, the secondary structure and dynamics of the recombinant protein corresponding to the intracellular region of Jagged-1, J1_tmic, bound to negatively charged lysophospholipid micelles. NMR showed that the PDZ binding motif is preceded by four alpha-helical segments and that, despite the extensive interaction between J1_tmic and the micelle, the PDZ binding motif remains highly flexible. Binding of J1_tmic to negatively charged, but not to zwitterionic vesicles, was confirmed by surface plasmon resonance. To study the PDZ binding region in more detail, we prepared a peptide corresponding to the last 24 residues of Jagged-1, J1C24, and different phosphorylated variants of it. J1C24 displays a marked helical propensity and undergoes a coil-helix transition in the presence of negatively charged, but not zwitterionic, lysophospholipid micelles. Phosphorylation at different positions drastically decreases the helical propensity of the peptides and abolishes the coil-helix transition triggered by lysophospholipid micelles. We propose that phosphorylation of residues upstream of the PDZ binding motif may shift the equilibrium from an ordered, membrane-bound, interfacial form of Jagged-1 C-terminal region to a more disordered form with an increased accessibility of the PDZ recognition motif, thus playing an indirect role in the interaction between Jagged-1 and the PDZ-containing target protein.

Jagged-1 juxtamembrane region: biochemical characterization and cleavage by ADAM17 (TACE) catalytic domain.

Ectodomain shedding of membrane receptors and ligands carried out by ADAMs (A disintegrin and metalloprotease) plays a major role in several signaling pathways, including Notch. The grounds of substrate recognition, however, are poorly understood. We demonstrate that a recombinant protein corresponding to the juxtamembrane region of Jagged-1, one of the Notch ligands, behaves as a structured module and is cleaved by ADAM17 catalytic domain at E1054. A short synthetic peptide is cleaved at the same site but at a much higher rate, implying that the structure of the cleavage site in the native protein is a key determinant for substrate recognition. We also show that an Alagille syndrome-associated mutation near E1054 increases the cleavage rate, which suggests that this mutation may lead to an unbalance in Notch signaling due to a higher level of Jagged-1 shedding.

Different degrees of structural order in distinct regions of the transcriptional repressor HES-1.

HES-1 is a transcriptional repressor of the basic helix-loop-helix (bHLH) family and one of the main downstream effectors in Notch signaling. Its domain architecture is composed of a bHLH region, an Orange domain, and a poorly characterized C-terminal half. We show that different degrees of structural order are present in the different regions of HES-1. The isolated bHLH domain is only marginally stable in solution, and partially folds upon dimerization. Binding to DNA promotes folding, stabilization, and protection from proteolysis of the bHLH domain. The Orange domain, on the contrary, is well folded in all conditions, forms stable dimers, and greatly increases protein resistance to thermal denaturation. The isolated proline-rich C-terminal region is mainly disordered in solution, and remains unstructured also in the full length protein. Measurements of binding constants show that HES-1 recognizes dsDNA synthetic oligonucleotides corresponding to several functional DNA targets with high affinity, but with relatively little specificity. We propose that order/disorder transitions in the different domains are associated not only with binding to DNA, but also with protein homo- and hetero-dimerization.

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 ((1213) RMEYIV(1218) ). 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.

Exon 6 of human JAG1 encodes a conserved structural unit.

BACKGROUND: Notch signaling drives developmental processes in all metazoans. The receptor binding region of the human Notch ligand Jagged-1 is made of a DSL (Delta/Serrate/Lag-2) domain and two atypical epidermal growth factor (EGF) repeats encoded by two exons, exon 5 and 6, which are out of phase with respect to the EGF domain boundaries. RESULTS: We determined the 1H-NMR solution structure of the polypeptide encoded by exon 6 of JAG1 and spanning the C-terminal region of EGF1 and the entire EGF2. We show that this single, evolutionary conserved exon defines an autonomous structural unit that, despite the minimal structural context, closely matches the structure of the same region in the entire receptor binding module. CONCLUSION: In eukaryotic genomes, exon and domain boundaries usually coincide. We report a case study where this assertion does not hold, and show that the autonomously folding, structural unit is delimited by exon boundaries, rather than by predicted domain boundaries.

Atom depth in protein structure and function.

Atom depth, originally defined as the distance between a protein atom and the nearest water molecule surrounding a protein, is a simple but valuable geometrical descriptor of the protein interior. It can be easily computed from the 3D structure of a protein, thus complementing the information provided by the calculation of the solvent accessible surface area and buried surface area. Depth has been found to be correlated with several molecular, residue and atomic properties, such as average protein domain size, protein stability, free energy of formation of protein complexes, amino acid type hydrophobicity, residue conservation and hydrogen/deuterium amide proton exchange rates.

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.

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 approximately 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.

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

The WWW servers at http://www.icgeb.org/protein/ 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.

Structure and dynamics of the anticodon arm binding domain of Bacillus stearothermophilus Tyrosyl-tRNA synthetase.

The structure of a recombinant protein, TyrRS(delta4), corresponding to the anticodon arm binding domain of Bacillus stearothermophilus tyrosyl-tRNA synthetase, has been solved, and its dynamics have been studied by nuclear magnetic resonance (NMR). It is the first structure described for such a domain of a tyrosyl-tRNA synthetase. It consists of a five-stranded beta sheet, packed against two alpha helices on one side and one alpha helix on the other side. A large part of the domain is structurally similar to other functionally unrelated RNA binding proteins. The basic residues known to be essential for tRNA binding and charging are exposed to the solvent on the same face of the molecule. The structure of TyrRS(delta4), together with previous mutagenesis data, allows one to delineate the region of interaction with tRNATyr.

The "first in-last out" hypothesis on protein folding revisited.

We calculated profiles for mean residue depth, contact order, and number of contacts in the native structure of a series of proteins for which folding has been studied extensively, the chymotrypsin inhibitor 2, the SH3 module from the src tyrosine kinase, the small ribonuclease barnase, the bacterial immunity protein Im7, and apomyoglobin. We compared these profiles with experimental data from equilibrium or pulse labeling hydrogen-deuterium exchange obtained from NMR and phi values obtained from the protein engineering approach. We find a good qualitative agreement between the hierarchy of formation of topological elements during the folding process and the ranking of secondary structure elements in terms of residue depth. Residues that are most deeply buried in the core of the native protein usually belong to stretches of secondary structure elements that are formed early in the folding pathway. Residue depth can thus provide a useful and simple tool for the design of folding experiments.

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.

Probing dynamic protein ensembles with atomic proximity measures.

The emerging role of internal dynamics in protein fold and function requires new avenues of structure analysis. We analyzed the dynamically restrained conformational ensemble of ubiquitin generated from residual dipolar coupling data, in terms of protruding and buried atoms as well as interatomic distances, using four proximity-based algorithms, CX, DPX, PRIDE and PRIDE-NMR (http://hydra.icgeb.trieste.it/protein/). We found that Ubiquitin, this relatively rigid molecule has a highly diverse dynamic ensemble. The environment of protruding atoms is highly variable across conformers, on the other hand, only a part of buried atoms tends to fluctuate. The variability of the ensemble cautions against the use of single conformers when explaining functional phenomena. We also give a detailed evaluation of PRIDE-NMR on a wide dataset and discuss its usage in the light of the features of available NMR distance restraint sets in public databases.

The tetralogy of Fallot-associated G274D mutation impairs folding of the second epidermal growth factor repeat in Jagged-1.

Notch signaling controls spatial patterning and cell-fate decisions in all metazoans. Mutations in JAG1, one of the five Notch ligands in man, have been associated with Alagille syndrome and with a familial form of tetralogy of Fallot. A specific G274D mutation in the second epidermal growth factor repeat of the Jagged-1 was found to correlate with tetralogy of Fallot symptoms but not with usual Alagille syndrome phenotypes. To investigate the effects of this mutation, we studied the in vitro oxidative folding of the wild-type and mutant peptides encompassing the second epidermal growth factor. We found that the G274D mutation strongly impairs the correct folding of the epidermal growth factor module, and folding cannot be rescued by compensative mutations. The 274 position displays very low tolerance to substitution because neither the G274S nor the G274A mutants could be refolded in vitro. A sequence comparison of epidermal growth factor repeats found in human proteins revealed that the pattern displayed by the second epidermal growth factor is exclusively found in Notch ligands and that G274 is absolutely conserved within this group. We carried out a systematic and comprehensive analysis of mutations found in epidermal growth factor repeats and show that specific residue requirements for folding, structural integrity and correct post-translational processing may provide a rationale for most of the disease-associated mutations.

Prevalence of intrinsic disorder in the intracellular region of human single-pass type I proteins: the case of the notch ligand Delta-4.

Intrinsic disorder (ID) is a widespread phenomenon found especially in signaling and regulation-related eukaryotic proteins. The functional importance of flexible disordered regions often resides in their ability to allow proteins to bind different partners. The incidence and location of intrinsic disorder in 369 human single-pass transmembrane receptors with the type I topology was assessed based on both disorder predictions and amino acid physico-chemical properties. We provide evidence that ID concentrates in the receptors' cytoplasmic region. As a benchmark for this analysis, we present a structural study on the previously uncharacterized intracellular region of human Delta-4 (DLL4_IC), a single-pass transmembrane protein and a ligand of Notch receptors. DLL4_IC is required for receptor/ligand endocytosis; it undergoes regulated intramembrane proteolysis, and mediates protein-protein interactions through its C-terminal PDZ binding motif. Using a recombinant purified protein, we demonstrate using various biophysical methods that DLL4_IC is mainly disordered in solution but can form interconvertible local secondary structures in response to variations in the physico-chemical milieu. Most of these conformational changes occur in the highly conserved C-terminal segment that includes the PDZ-binding motif. On the basis of our results, we propose that global disorder, in concert with local preorganization, may play a role in Notch signaling mediated by Delta-4.

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

The 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.

Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region.

Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purification, and characterization of the intracellular region of a Notch ligand. Starting from a set of synthetic oligonucleotides, we assembled a synthetic gene optimized for Escherichia coli codon usage and encoding the cytoplasmic region of human Jagged-1 (residues 1094-1218). The protein containing a N-terminal His(6)-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His(6)-tag by a dipeptidyl aminopeptidase, the protein was purified to homogeneity and characterized by spectroscopic techniques. Far-UV circular dichroism, fluorescence emission spectra, fluorescence anisotropy measurements, and (1)H nuclear magnetic resonance spectra, taken together, suggest that the cytoplasmic tail of human Jagged-1 behaves as an intrinsically unstructured domain in solution. This result was confirmed by the high susceptibility of the recombinant protein to proteolytic cleavage. The significance of this finding is discussed in relation to the recently proposed role of the intracellular region of Notch ligands in bi-directional signaling.

Efficient synthesis and comparative studies of the arginine and Nomega,Nomega-dimethylarginine forms of the human nucleolin glycine/arginine rich domain.

The Gly- and Arg-rich C-terminal region of human nucleolin is a 61-residue long domain involved in a number of protein-protein and protein-nucleic acid interactions. This domain contains 10 aDma residues in the form of aDma-GG repeats interspersed with Phe residues. The exact role of Arg dimethylation is not known, partly because of the lack of efficient synthetic methods. This work describes an effective synthetic strategy, generally applicable to long RGG peptides, based on side-chain protected aDma and backbone protected dipeptide Fmoc-Gly-(Dmob)Gly-OH. This strategy allowed us to synthesize both the unmodified (N61Arg) and the dimethylated (N61aDma) peptides with high yield ( approximately 26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and N(omega),N(omega)-dimethylation of the guanidino group does not alter the overall conformational propensity of this peptide. While both peptides bind single-stranded nucleic acids with similar affinities (K(d) = 1.5 x 10(-7) M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pK(a) values. It has been previously shown that N61Arg inhibits HIV infection at the stage of HIV attachment to cells. This study demonstrates that Arg-dimethylated C-terminal domain lacks any inhibition activity, raising the question of whether nucleolin expressed on the cell-surface is indeed dimethylated.

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.