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 ...

A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa (EC 3.4.21.21), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation with zymogen-like properties. Association with tissue factor (TF), locally exposed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by replacing residues believed to function as determinants for the inherent zymogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appear to retain the substrate specificity of the parent enzyme and can be further stimulated by TF. Insights into the mechanism behind the increased activity of the mutants, presumably also pertinent to the TFinduced, allosteric stimulation of FVIIa activity, were obtained by studying their calcium dependence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficacious treatment of bleeding episodes especially in hemophiliacs with inhibitory antibodies precluding conventional replacement therapy.

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.

Factor VIIa (EC 3.4.21.21) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade. On injury, factor VIIa forms a complex with its allosteric regulator, tissue factor, and initiates blood clotting. More importantly, a surface-exposed ␣-helix in the protease domain (residues 307-312), which is located at the cofactor recognition site, is distorted in the free form of factor VIIa. This subtle structural difference sheds light on the mechanism of the dramatic tissue factor-induced enhancement of factor VIIa activity.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TFbound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50 Å and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases. Coagulation factor VII (FVII)2 circulates in the blood with ϳ1% in a two-chain form (FVIIa) and the remainder as singlechain zymogen FVII. FVIIa consists of a trypsin-like protease domain and an N-terminal light chain composed of a membrane-binding ␥-carboxyglutamate-rich domain (Gla domain) and two epidermal growth factor-like domains (EGF1 and EGF2) (1). Upon tissue injury, tissue factor (TF) becomes exposed, and the TF⅐FVIIa complex forms and serves as the initiator of the blood coagulation cascade (2). Numerous coagulation proteases function optimally only when complexed to cofactors, and substantial biochemical evidence supports the concept that several of these cofactors induce allosteric changes in the conformation of their cognate enzymes (1, 2). TF functions to localize FVIIa and as an allosteric regulator, which dramatically enhances the activity of FVIIa. Similar to the trypsinogen-trypsin pair, zymogen FVII is converted to FVIIa by proteolysis of an internal peptide bond. A canonical "activation domain" was defined in trypsin, which includes the N terminus created upon endoproteolytic activation and three loops referred to as the activation loops (3). In trypsin, the newly generated N-terminal tail spontaneously inserts itself into a cavity close to the three activation loops, termed the activation pocket, resulting in the formation of a critical salt bridge between the N-terminal Ile-16 and Asp-194 of the active site or and in FVIIa (the chymotrypsin numbering is denoted in superscript with parentheses). This salt bridge leads to the formation of a correctly assembled S1 pocket of the active site and full activity. FVIIa, however, has very low activity following endoproteolytic activation and does not spontaneously rearrange into the active form. The three-dimensional structure of FVIIa bound to TF has been solved providing important structural details of the complex and the active form of FVIIa (4). In the complex, parts of the light chain a...

To evaluate the possible relationship between biological activity and structural stability in selected regions of the insulin molecule, we have analyzed the guanidine hydrochloride induced reversible unfolding of a series of mutant insulins using a combination of near- and far-UV circular dichroism (CD). The unfolding curves are reasonably described on the basis of a two-state denaturation scheme; however, the observation of subtle differences between near- and far-UV CD detected unfolding indicates that intermediates may be present. Three regions of the insulin molecule are analyzed in detail with respect to their contribution to folding stability, i.e., the central B-chain helix, the NH2-terminal A-chain helix, and the B25-B30 extended chain region. Considerable enhancement of folding stability is engineered by mutations at the N-cap of the central B-chain helix and at the C-cap of the NH2-terminal A-chain helix. Mutations that confer increased stability in these regions are identical to those that lead to enhanced biological activity. In contrast, for insulin species modified in the B25-B30 region of the molecule, we observe no correlation between global folding stability and bioactivity. Mutations in the three regions examined are found to affect stability in a nearly independent fashion, and stabilizing mutations are generally found to enhance the cooperativity of the unfolding transition. We conclude that highly potent insulins (i.e., HisA8, ArgA8, GluB10, and AspB10) elicit enhanced activity because these mutations stabilize structural motifs of critical importance for receptor recognition.

The enzyme factor VIIa (FVIIa) triggers the blood coagulation cascade upon association with tissue factor (TF). The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex, and Met-306 in the serine protease domain of FVIIa participates in this event. We have characterized FVIIa variants mutated in position 306 with respect to their ability to be stimulated by TF. The amidolytic activity of FVIIa mutants with Ser, Thr, and Asn in position 306 was stimulated 9-, 12-, and 7-fold, respectively, by soluble TF as compared to 22-fold for wild-type FVIIa. In contrast, the activity of Met306Asp-FVIIa only increased about 2-fold and that of Met306Asp/Asp309Ser-FVIIa increased about 1.5-fold. Modeling suggests that Asp in position 306 prevents the TF-induced stimulation of FVIIa by disrupting essential intermolecular hydrogen bonds. The ability of the FVIIa variants to catalyze factor X activation and the amidolytic activity were enhanced to a similar extent by soluble TF. This indicates that factor X does not promote its own activation through interactions with exosites on FVIIa made accessible upon FVIIa-TF assembly. Met306Asp-FVIIa binds soluble TF with a dissociation constant of 13 nM (about 3-fold higher than that of FVIIa), and, in sharp contrast to FVIIa, its binding kinetics are unaltered after inactivation with D-Phe-Phe-Arg chloromethyl ketone. We conclude that a single specific amino acid replacement, substitution of Asp for Met-306, virtually prevents the TF-induced allosteric changes which normally result in dramatically increased FVIIa activity and eliminates the effect of the active site inhibitor on TF affinity.