With the current access to the whole genomes of various organisms and the completion of the first draft of the human genome, there is a strong need for a structure-function classification of protein families as an initial step in moving from DNA databases to a comprehensive understanding of human biology. As a result of the explosion in nucleic acid sequence information and the concurrent development of methods for high-throughput functional characterization of gene products, the genomic revolution also promises to provide a new paradigm for drug discovery, enabling the identification of molecular drug targets in a significant number of human diseases. This molecular view of diseases has contributed to the importance of combining primary sequence data with three-dimensional structure and has increased the awareness of computational homology modeling and its potential to elucidate protein function. In particular, when important proteins or novel therapeutic targets are identified-like the family of protein tyrosine phosphatases (PTPs) (reviewed in reference 53)-a structure-function classification of such protein families becomes an invaluable framework for further advances in biomedical science. Here, we present a comparative analysis of the structural relationships among vertebrate PTP domains and provide a comprehensive resource for sequence analysis of phosphotyrosine-specific PTPs.PTPs are a key group of signal transduction enzymes which, together with protein tyrosine kinases, control the levels of cellular protein tyrosine phosphorylation. Protein tyrosine kinases phosphorylate cellular substrates on tyrosine residues, and much progress has been made over the last 20 years in elucidating their significance in signal transduction (for reviews, see references 26, 30, 31, 33, 71, and 72). However, it is only recently that the complexities of the PTPs have been appreciated. Thus, today it is recognized that the capacity of PTPs to dephosphorylate phosphotyrosine residues selectively on their substrates plays a pivotal role in initiating, sustaining and terminating cellular signaling (for reviews, see references 1, 4, 19, 32, 35, 46, 55, and 83). It has been shown that both the catalytic domain and noncatalytic segments of the PTPs contribute to the definition of substrate specificity in vivo. Whereas noncatalytic domains may target the PTPs to specific intracellular compartments in which the effective local concentration of substrate is high (3, 19, 51), the PTP catalytic domains themselves confer site-selective protein dephosphorylation by recognizing both the phosphotyrosine residue to be dephosphorylated and its flanking amino acids in the substrate. The combination of structural studies, kinetic analysis of PTP domains (37,74,76,90,91,96), and studies involving substratetrapping mutants (20,23,89) as well as PTP chimeras (60, 82) has convincingly demonstrated that isolated PTP domains may exhibit exquisite substrate selectivity.The structurally conserved PTP domain defines membership of the PTP family, and ...

Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory disease causes increased vascular permeability and edema formation through unknown mechanisms. Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration in endothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining, leukocytic beta2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/azurocidin, a member of the serprocidin family of neutrophil cationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvessels in vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducing endothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derived HBP in the vascular response to neutrophil trafficking in inflammation. Targeting this molecule in inflammatory disease conditions offers a new strategy for prevention of endothelial barrier dysfunction caused by misdirected leukocyte activation.

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization K d is measured to be ∼1 × 10 3 molecules/μm 2 , and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.Ras signaling | Ras assay

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane curvature was essential for enrichment in raft-like liquid-ordered phases; enrichment was driven by relief of lateral pressure upon anchor insertion and most likely affects the localization of lipidated proteins in general.

Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130 -132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme.Protein-tyrosine phosphatases (PTPs) 1 are key regulators of signal transduction processes (1, 2). The family of classical PTPs can be divided into two broad categories as intracellular and receptor-like PTPs covering a total of 17 subtypes (3). Receptor-like PTPs contain an extracellular domain, a single transmembrane domain, and one or two cytoplasmic PTP domains. Intracellular PTPs generally contain one PTP domain and an N-or C-terminal domain that targets the enzymes to specific subcellular localizations, as exemplified by the targeting of PTP1B to the endoplasmic reticulum (4).PTP1B and TC-PTP are two closely related intracellular enzymes. PTP1B was the first protein-tyrosine phosphatase to be identified and characterized (5, 6). Shortly after this landmark event, PTP1B was cloned from a placenta cDNA library (7), and TC-PTP was cloned from a peripheral human T cell cDNA library (8). Despite its name, TC-PTP is ubiquitously expressed (9). Alternative splicing gives rise to two forms of TC-PTP that differ in the C termini, a 45-kDa form that is targeted to the nucleus and a 48-kDa form that localizes to the endoplasmic reticulum via a hydrophobic C-terminal region (10). TC-PTP is tightly regulated during the cell cycle and seems to play an important role in mitogenesis (9). In a recent study, it was shown that cellular stress causes reversible cytoplasmic accumulation of the 45-kDa form of TC-PTP (i.e. the nuclear form) (11).Although they have a sequence identity of about 74% in the catalytic domains (see Fig. 1), TC-PTP and PTP1B clearly fulfill different biological functions, as has been demonstrated in knock-out mice. Thus, although PTP1B knock-out mice show increased insulin sensitivity and resistance to diet-induced obesity and are viable with a normal life span (12, 13), TC-PTP knock-out mice die at 3-5 weeks of age (14).In accordance with these in vivo observations, substrate trapping experiment...

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp 181 , Lys 120 , and Tyr 46 . PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.