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Biomedical subjects

O H Olsen

Publications and source records attributed to O H Olsen.

At least 19 recordsLinked to original sources
PubMed2001

Steric hindrance as a basis for structure-based design of selective inhibitors of protein-tyrosine phosphatases.

Utilizing structure-based design, we have previously demonstrated that it is possible to obtain selective inhibitors of protein-tyrosine phosphatase 1B (PTP1B). A basic nitrogen was introduced into a general PTP inhibitor to form a salt bridge to Asp48 in PTP1B and simultaneously cause repulsion in PTPs containing an asparagine in the equivalent position [Iversen, L. F., et al. (2000) J. Biol. Chem. 275, 10300-10307]. Further, we have recently demonstrated that Gly259 in PTP1B forms the bottom of a gateway that allows easy access to the active site for a broad range of substrates, while bulky residues in the same position in other PTPs cause steric hindrance and reduced substrate recognition capacity [Peters, G. H., et al. (2000) J. Biol. Chem. 275, 18201-18209]. The current study was undertaken to investigate the feasibility of structure-based design, utilizing these differences in accessibility to the active site among various PTPs. We show that a general, low-molecular weight PTP inhibitor can be developed into a highly selective inhibitor for PTP1B and TC-PTP by introducing a substituent, which is designed to address the region around residues 258 and 259. Detailed enzyme kinetic analysis with a set of wild-type and mutant PTPs, X-ray protein crystallography, and molecular modeling studies confirmed that selectivity for PTP1B and TC-PTP was achieved due to steric hindrance imposed by bulky position 259 residues in other PTPs.

Animals↗
PubMed2001

Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.

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 TF-induced, 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.

Antithrombins↗
PubMed2001

Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity.

Factor VII requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active factor VIIa (FVIIa) conformation. The former event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. We have designed a number of FVIIa mutants with the aim of mimicking the effect of TF, that is, creating molecules with increased intrinsic (TF-independent) enzymatic activity. Based on a possible structural difference between free and TF-bound FVIIa (Pike, A. C. W., Brzozowski, A. M., Roberts, S. M., Olsen, O. H., and Persson, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8925--8930), we focused on the helical region encompassing residues 307-312 and residues in its spatial vicinity. For instance, FVIIa contains Phe-374 and Leu-305, whereas a Phe/Tyr residue in the position corresponding to 374 in homologous coagulation serine proteases is accompanied by Val in the position corresponding to 305. This conceivably results in a unique orientation of this helix in FVIIa. Substitution of Val for Leu-305 in FVIIa resulted in a 3--4-fold increase in the intrinsic amidolytic and proteolytic activity as compared with wild-type FVIIa, whereas the activity in complex with soluble TF remained the same. In accordance with this, L305V-FVIIa exhibited an increased rate of inhibition as compared with wild-type FVIIa, both by d-Phe-Phe-Arg-chloromethyl ketone and antithrombin III in the presence of heparin. The increased FVIIa activity upon replacement of Leu-305 by Val may be mediated by a movement of the 307--312 helix into an orientation resembling that found in factors IXa and Xa and thrombin. The corresponding shortening of the side chain of residue 374 (Phe --> Pro) had a smaller effect (about 1.5-fold increase) on the intrinsic activity of FVIIa. Attempts to increase FVIIa activity by introducing single or multiple mutations at positions 306, 309, and 312 to stabilize the 307-312 helix failed.

Amino Acid Substitution↗
PubMed2001

Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor.

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.

Allosteric Regulation↗
PubMed2000

Residue 259 is a key determinant of substrate specificity of protein-tyrosine phosphatases 1B and alpha.

The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine phosphatases (PTPs), PTP1B and PTPalpha, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH(2) peptide is well recognized by PTP1B, but less efficiently by PTPalpha, it was chosen as a tool for these analyses. Calpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity-determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTPalpha, either in combination or alone, we here demonstrate that the key selectivity-determining residue is 259. In PTPalpha, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln(259) with a glycine almost turns PTPalpha into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln(259) in PTPalpha plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln(262)). Both effects may indicate that PTPalpha regulates highly selective signal transduction processes.

Animals↗
PubMed2000

Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B.

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.

Animals↗
PubMed2000

Molecular dynamics simulations of protein-tyrosine phosphatase 1B. II. substrate-enzyme interactions and dynamics.

Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme and substrate flexibility for binding, 3) the electrostatic properties of the enzyme, and 4) the contribution from solvation. The simulations were performed for 1 ns, using explicit water molecules. The last 700 ps of the trajectories was used for analysis determining enthalpic and entropic contributions to substrate binding. Based on essential dynamics analysis of the PTP1B/DADEpYL trajectory, it is shown that internal motions in the binding pocket occur in a subspace of only a few degrees of freedom. In particular, relatively large flexibilities are observed along several eigenvectors in the segments: Arg(24)-Ser(28), Pro(38)-Arg(47), and Glu(115)-Gly(117). These motions are correlated to the C- and N-terminal motions of the substrate. Relatively small fluctuations are observed in the region of the consensus active site motif (H/V)CX(5)R(S/T) and in the region of the WPD loop, which contains the general acid for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein surface is characterized by a predominantly negative electrostatic field. This positive electrostatic field attracts negatively charged substrates and could explain the experimentally observed preference of PTP1B for negatively charged substrates like the DADEpYL peptide.

Amino Acid Sequence↗
PubMed2000

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor.

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.

Alanine↗
PubMed1999

Structure of human factor VIIa and its implications for the triggering of blood coagulation.

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. Although the structure of the binary complex has already been determined [Banner, D. W., D'Arcy, A., Chène, C., Winkler, F. K., Guha, A., Konigsberg, W. H., Nemerson, Y. & Kirchhofer, D. (1996) Nature (London) 380, 41-46], the conformational effects of cofactor binding to factor VIIa are not known in detail because of a lack of structural information on free factor VIIa. Here we report the structure of gamma-carboxyglutamic acid-domainless human coagulation factor VIIa at a resolution of 2.8 A. The molecule adopts an extended conformation within the crystal similar to that previously observed for the full-length protein in complex with tissue factor. Detailed comparison of free and tissue factor-bound factor VIIa reveals several structural differences. The binding mode of the active-site inhibitor D-Phe-Phe-Arg methyl ketone differs in the two structures, suggesting a role for the cofactor in substrate recognition. More importantly, a surface-exposed alpha-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.

Amino Acid Sequence↗
PubMed1999

Molecular dynamics simulations of protein-tyrosine phosphatase 1B. I. ligand-induced changes in the protein motions.

Activity of enzymes, such as protein tyrosine phosphatases (PTPs), is often associated with structural changes in the enzyme, resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate on the motions occurring in PTPs, we have performed molecular dynamics simulations of PTP1B and PTP1B complexed with a high-affinity peptide DADEpYL, where pY stands for phosphorylated tyrosine. The peptide sequence is derived from the epidermal growth factor receptor (EGFR988-993). Simulations were performed in water for 1 ns, and the concerted motions in the protein were analyzed using the essential dynamics technique. Our results indicate that the predominately internal motions in PTP1B occur in a subspace of only a few degrees of freedom. Upon substrate binding, the flexibility of the protein is reduced by approximately 10%. The largest effect is found in the protein region, where the N-terminal of the substrate is located, and in the loop region Val198-Gly209. Displacements in the latter loop are associated with the motions in the WPD loop, which contains a catalytically important aspartic acid. Estimation of the pKa of the active-site cysteine along the trajectory indicates that structural inhomogeneity causes the pKa to vary by approximately +/-1 pKa unit. In agreement with experimental observations, the active-site cysteine is negatively charged at physiological pH.

Algorithms↗
PubMed1999

Properties of spin and fluorescent labels at a receptor-ligand interface.

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.

Binding Sites↗
PubMed1999

Assignment of side-chain conformation using adiabatic energy mapping, free energy perturbation, and molecular dynamic simulations.

NMR spectroscopic analysis of the C-terminal Kunitz domain fragment (alpha3(VI)) from the human alpha3-chain of type VI collagen has revealed that the side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X-ray crystallographic structure, while the minor conformation (m) cannot structurally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conformations of the Trp21 side chain obtained by energy minimization identified two energy minima: One corresponding to the conformation of Trp21 observed in the X-ray crystallographic structure and solution structure of alpha3(VI) (the M conformation) and the second corresponding to the m conformation predicted by NMR spectroscopy. A transition pathway between the M and m conformation is suggested. The free-energy difference between the two conformers obtained by the thermodynamic integration method is calculated to 1.77+/-0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformers of the alpha3(VI) molecule were investigated by molecular dynamic. Essential dynamics analysis of the two resulting 800 ps trajectories reveals that when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are observed in the mobility of the binding loop (residues Thr13-Ile18), which is more flexible in the m conformation than in the M conformation. This suggests that the reorientation of Trp2 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding loop and Trp21.

Collagen↗
PubMed1998

Electrostatic evaluation of the signature motif (H/V)CX5R(S/T) in protein-tyrosine phosphatases.

The catalytic activity of protein-tyrosine phosphatases (PTPs) is mediated by a cysteine side chain which carries out a nucleophilic attack initiating the phosphate cleavage. Experimentally, it has been observed that the active site cysteine has a remarkably low pKa. In the present study, we have investigated the origin of the low pKa by analyzing the electrostatic properties of four different protein-tyrosine phosphatases: Yersinia PTP (bacteria), PTP1B (human), VHR (human), and low molecular weight phosphatase (bovine). These phosphatases have very low sequence homology and show very low structural similarity. However, they share a common active site motif [the (H/V)CX5R(S/T) sequence] which adopts a unique loop structure. We have applied the so-called single site titration method, which is based on the Poisson-Boltzmann methodology, to (i) study the influence of the architecture of the (H/V)CX5R(S/T) loop on the pKa of the active cysteine and (ii) examine which parts of the active site region stabilize the ionized form of the cysteine. Our results indicate that the architecture of the (H/V)CX5R(S/T) loop has a major impact on the low pKa of the active cysteines. The orientation of the microdipoles generated by the partial charges of the backbone atoms (i.e., the CONHCalpha atoms) is essential for maintaining the low pKa. Further, the electrostatic field generated by these microdipoles has a larger impact than the electrostatic dipole generated by the central alpha-helix. Interactions of the active cysteine with other ionizable side chains play a minor role in stabilizing the thiolate anion. The only ionizable side chain significantly influencing the pKa of the active site cysteine is the arginine, which is an important part of the consensus sequence.

Amino Acid Sequence↗
PubMed1998

13C NMR, X-ray, and differential scanning calorimetry investigations of truncated BPTI (aprotinin) analogues.

Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other residues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variations for the backbone, but the lack of salt bridge between the terminals and the lack of two N-terminal residues provide a structure open at one end. Comparisons of amide exchange rates show a dramatic increase for the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wild-type despite only small differences in the structures. The amide exchange rates for truncated analogues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to 13C chemical shifts are made. 13C chemical shifts are assigned using the D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. 13C NMR chemical shifts are shown to be able to gauge structural changes. A comparison of 13C chemical shifts of WT BPTI (aprotinin) and 3-58BPTI reveals effects caused by (i) the removal of the salt bridge of the terminii, (ii) the charge of the N-terminus, and (iii) the increased mobility of the side chain of Tyr-23. Small effects are also seen due to a conformational change of the aromatic ring of Phe-4. Ring current shifts at 13C chemical shifts are calculated. The difference in the calculated ring current effects are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) provided the structures are relaxed. Protein unfolding as a function of pH and temperature is studied by DSC. Unfolding occurs at lower temperature with N-terminally truncated analogues, and the maximum is shifted toward higher pH.

Amino Acid Substitution↗
PubMed1997

Ca2+ binding to the first epidermal growth factor-like domain of factor VIIa increases amidolytic activity and tissue factor affinity.

Coagulation factor VIIa belongs to a family of homologous enzymes, including factors IXa and Xa and activated protein C, composed of two epidermal growth factor-like domains located between an N-terminal domain rich in gamma-carboxyglutamic acid residues and a C-terminal serine protease domain. The first epidermal growth factor-like domain in factor VIIa contains a Ca2+ binding site, the function of which is largely unknown. Site-directed mutagenesis of two Ca2+-liganding Asp residues in this domain abolished Ca2+ binding and resulted in a 2-3-fold decrease in amidolytic activity at optimal Ca2+ concentrations. The lower amidolytic activity persisted in complex with soluble tissue factor, apparently due to a lower kcat of the mutant factor VIIa. Mutant and wild-type factor VIIa bound to lipidated tissue factor were equally efficient activators of factor X. The dissociation constants, derived from amidolytic activity and surface plasmon resonance measurements, were 2-5 nM and 50-60 nM for the interactions between wild-type and mutant factor VIIa, respectively, and soluble tissue factor. Binding to lipidated tissue factor was characterized by dissociation constants of 7.5 pM for factor VIIa and 160 pM for the factor VIIa mutant. Hence, a functional Ca2+ binding site in the first epidermal growth factor-like domain added 7-8 kJ/mol to the total binding energy of the interaction with both lipidated and soluble tissue factor.

Aspartic Acid↗