Biomedical subjects

J A Huntington

Publications and source records attributed to J A Huntington.

At least 19 recordsLinked to original sources

PubMed2003

Mechanisms of glycosaminoglycan activation of the serpins in hemostasis.

Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final serpin-protease complex. The serpin mechanism, in contrast to the classical lock-and-key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and alpha2-antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the serpin mechanism has been fine-tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.

Animals↗

PubMed2001

NTF2 monomer-dimer equilibrium.

Nuclear transport factor 2 (NTF2) mediates nuclear import of RanGDP, a central component of many nuclear trafficking pathways. NTF2 is a homodimer and each chain has independent binding sites for RanGDP and nuclear pore proteins (nucleoporins) that contain FxFG sequence repeats. We show here that the monomer-dimer dissociation constant for NTF2 obtained by sedimentation equilibrium ultracentrifugation is in the micromolar range, indicating that a substantial proportion of cellular NTF2 may be monomeric. To investigate the functional significance of NTF2 dimerization, we engineered a series of point mutations at the dimerization interface and one of these (M118E) remained monomeric below concentrations of 150 microM. CD spectra and X-ray crystallography showed that M118E-NTF2 preserved the wild-type NTF2 fold, although its thermal stability was 20 deg. C lower than that of the wild-type. M118E-NTF2 bound both RanGDP and FxFG nucleoporins less strongly, suggesting that dissociation of the NTF2 dimer could facilitate RanGDP release and thus nucleotide exchange after it had been transported into the nucleus. Moreover, colloidal gold coated with M118E-NTF2 showed reduced binding to Xenopus oocyte nuclear pores. Overall, our results indicate that dimer formation is important for NTF2 function and give insight into the formation of heterodimers by mRNA export factors such as TAP1 and NXT1 that contain NTF2-homology domains.

Amino Acid Substitution↗

PubMed2001

Structure and properties of ovalbumin.

Ovalbumin is a protein of unknown function found in large quantities in avian egg-white. Surprisingly, ovalbumin belongs to the serpin family although it lacks any protease inhibitory activity. We review here what is known about the amino acid sequence, post-translational modifications and tertiary structure of ovalbumin. The properties of ovalbumin are discussed in relation to their possible functional significance. These include reasons for failure of ovalbumin to undergo a typical serpin conformational change involving the reactive centre loop, which explains why ovalbumin is not a protease inhibitor, and also the natural conversion of ovalbumin to the more stable "S" form.

Amino Acid Sequence↗

PubMed2001

The serpin inhibitory mechanism is critically dependent on the length of the reactive center loop.

The recent crystallographic structure of a serpin-protease complex revealed that protease inactivation results from a disruption of the catalytic site architecture caused by the displacement of the catalytic serine. We hypothesize that inhibition depends on the length of the N-terminal portion of the reactive center loop, to which the active serine is covalently attached. To test this, alpha(1)-antitrypsin Pittsburgh variants were prepared with lengthened and shortened reactive center loops. The rates of inhibition of factor Xa and of complex dissociation were measured. The addition of one residue reduced the stability of the complex more than 200,000-fold, and the addition of two residues reduced it by more than 1,000,000-fold, whereas the deletion of one or two residues lowered the efficiency of inhibition and increased the stability of the complex (2-fold). The deletion of more than two residues completely converted the serpin into a substrate. Similar results were obtained for the alpha(1)-antitrypsin variants with thrombin and for PAI-1 and PAI-2 with their common target tissue plasminogen activator. We conclude that the length of the serpin reactive center loop is critical for its mechanism of inhibition and is precisely regulated to balance the efficiency of inhibition and stability of the final complex.

Amino Acid Sequence↗

PubMed2001

The serpins: nature's molecular mousetraps.

A special family of inhibitors, known as the serpins, has evolved an extraordinary mechanism to enable the control of the proteolytic pathways essential to life. The serpins undergo a profound change in conformation to entrap their target protease in an irreversible complex. The solving of the structure of this complex now completes a video depiction of the changes involved. The serpin, just like a mousetrap, is seen to change with a spring-like movement from an initial metastable state to a final hyperstable form. The structure shows how this conformational shift not only inhibits the protease but also destroys it. A bonus from these structural insights is the realisation that a number of diseases, as diverse as thrombosis, cirrhosis and dementia, all share a common mechanism arising from similar mutations of different serpins.

Animals↗

PubMed2001

The conformational basis of thrombosis.

Antithrombin readily undergoes a spontaneous transition from its active five-stranded form to a six-stranded inactive latent form. The recognition of this change in plasma has been obscured by the immediate linkage of newly formed latent antithrombin to a molecule of active antithrombin to give a dimer with an electrophoretic mobility readily confused with that of native active antithrombin. A new micromethod now allows unequivocal identification of latent antithrombin in whole plasma. This shows that at 37 degrees C some 10% of plasma antithrombin is converted to the latent form in 24 h. The rate of conversion is greatly accelerated at increased temperatures, as occurs in the pasteurisation of plasma concentrates that should now be checked for efficacy. But increased transition also occurs in the plasma at the slightly increased temperatures that accompany incidental infections. This is of particular significance if there is a conformationally unstable variant of antithrombin; here fever can provoke a sudden transition with the onset of a characteristically severe episode of thromboembolism. Such variants are not rare and include those previously classified as pleiotropic. The precise structural pathway, now known with antithrombin, provides a model of the changes occurring in other conformational diseases, including Alzheimer's and the prion dementias.

Antithrombins↗

PubMed2000

Structure of a serpin-protease complex shows inhibition by deformation.

The serpins have evolved to be the predominant family of serine-protease inhibitors in man. Their unique mechanism of inhibition involves a profound change in conformation, although the nature and significance of this change has been controversial. Here we report the crystallographic structure of a typical serpin-protease complex and show the mechanism of inhibition. The conformational change is initiated by reaction of the active serine of the protease with the reactive centre of the serpin. This cleaves the reactive centre, which then moves 71 A to the opposite pole of the serpin, taking the tethered protease with it. The tight linkage of the two molecules and resulting overlap of their structures does not affect the hyperstable serpin, but causes a surprising 37% loss of structure in the protease. This is induced by the plucking of the serine from its active site, together with breakage of interactions formed during zymogen activation. The disruption of the catalytic site prevents the release of the protease from the complex, and the structural disorder allows its proteolytic destruction. It is this ability of the conformational mechanism to crush as well as inhibit proteases that provides the serpins with their selective advantage.

Animals↗

PubMed2000

The conformational activation of antithrombin. A 2.85-A structure of a fluorescein derivative reveals an electrostatic link between the hinge and heparin binding regions.

Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined the crystal structure of an activated antithrombin variant, N135Q S380C-fluorescein (P14-fluorescein), in order to see how full activation is achieved in the absence of heparin and how the structural effects of the substitution in the hinge region are translated to the heparin binding region. The crystal structure resembles native antithrombin except in the hinge and heparin binding regions. The absence of global conformational change allows for identification of specific interactions, centered on Glu(381) (P13), that are responsible for maintenance of the solution equilibrium between the native and activated forms and establishes the existence of an electrostatic link between the hinge region and the heparin binding region. A revised model for the mechanism of the allosteric activation of antithrombin is proposed.

Amino Acid Sequence↗

PubMed2000

The effect of a reducing-end extension on pentasaccharide binding by antithrombin.

Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific pentasaccharide domain in a two-step mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHG'H'). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths >/=0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.

Antithrombins↗

PubMed1999

Formation of the antithrombin heterodimer in vivo and the onset of thrombosis.

Antithrombin is shown to undergo a slow spontaneous conversion to its inactive latent conformation with readily discernible amounts present in plasma on incubation at 37 degrees C for 72 hours. More rapid conversion occurs on incubation of isolated antithrombin at 41 degrees C or 50 degrees C, but the appearance on electrophoresis of free latent antithrombin is preceded by the formation, in reciprocal proportions, of a new slow band. This slow component is shown to be a heterodimer of active and latent antithrombin. It can be isolated as a single stable band either by incubation of antithrombin or by mixing equimolar proportions of active and latent antithrombin under the same conditions that give overnight crystallization of the active/latent antithrombin heterodimer. Similarly, equimolar addition of latent antithrombin to plasma results electrophoretically in a quantitative shift to the slower heterodimer mobility. Clinically, the presence of latent antithrombin is potentially deleterious, because its linkage to form the heterodimer results in inactivation of the otherwise normal molecule linked to the latent antithrombin. In the case of alpha-antithrombin, because the dimer readily dissociates, there is only a 11% additive loss of activity, but with beta-antithrombin the dimer appears more stable, with the additive loss of activity from the normal beta component being 21%, increasing to 33% on stabilization of the dimer with heparin. This linked and selective loss of activity of beta-antithrombin provides an explanation for the unexpected severity of thrombotic episodes in heterozygotes with conformationally unstable antithrombins.

Antithrombins↗

PubMed1999

A 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.

Amino Acid Sequence↗

PubMed1998

Conformational conversion of antithrombin to a fully activated substrate of factor Xa without need for heparin.

Regulation of the inhibitory activity of antithrombin, the principal inhibitor of the blood-clotting proteinases factor Xa and thrombin, is accomplished by binding to heparin. We report here an antithrombin variant in which serine at position 380, 14 residues N-terminal from the reactive bond and at a hinge point in the structure, was replaced by cysteine to test a proposed mechanism of heparin activation of antithrombin as an inhibitor of factor Xa. By derivatization of this cysteine with a bulky group, fluorescein, the antithrombin became permanently and fully activated toward reaction with factor Xa in a manner analogous to heparin activation, albeit as a substrate. These findings establish a structural basis for the mechanism of heparin activation of antithrombin against factor Xa in agreement with that proposed from an X-ray structure of antithrombin.

Animals↗

PubMed1998

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Amino Acid Sequence↗

PubMed1997

The P6-P2 region of serpins is critical for proteinase inhibition and complex stability.

Two of the prototypic serpins are alpha1-proteinase inhibitor and ovalbumin. alpha1-Proteinase inhibitor is a rapid inhibitor of a number of proteinases and undergoes the characteristic serpin conformational change on cleavage within the reactive center loop, whereas ovalbumin is noninhibitory and does not undergo the conformational change. To investigate if residues from P12 to P2 in the reactive center loop of ovalbumin are intrinsically incapable of being in an inhibitory serpin, we have made chimeric alpha1-proteinase inhibitor variants containing residues P12-P7, P6-P2, or P12-P2 of ovalbumin and determined their inhibitory properties with trypsin and human neutrophil elastase. With the P12-P7 and P6-P2 variants, the steps before and after the fork of the branched suicide-substrate pathway were affected as reflected by changes in rates and stoichiometries of inhibition with both proteinases. The P12-P2 variant showed that those effects were nonadditive, with exclusive substrate behavior for elastase and only residual inhibitory activity against trypsin. The properties of the variants were consistent with them obeying the suicide-substrate mechanism characteristic of serpins. Enzyme activity was regenerated from complexes formed with the P6-P2 variant faster than with wild-type indicating that the rate of turnover of the complex was increased. Based on proteinase susceptibility in the reactive center loops of the P6-P2 and P12-P2 variants, and on an increase in heat stability of the cleaved P12-P2 variant, it was concluded that the variants had undergone complete loop insertion on cleavage. These results show that the reactive center loop residues P12-P2 of ovalbumin can be present in inhibitory serpins although decreasing the inhibitory properties. These data also demonstrate that the residues in the P6-P2 region of serpins are critical for rapid inhibition of proteinases and formation of stable serpin-proteinase complexes.

Amino Acid Sequence↗

PubMed1997

Serpin conformational change in ovalbumin. Enhanced reactive center loop insertion through hinge region mutations.

Ovalbumin is a noninhibitory member of the serpin superfamily that does not spontaneously undergo the loop-to-sheet conformational change upon cleavage of its reactive center that is characteristic of inhibitory serpins. We tested the hypothesis that ovalbumin could be turned into a proteinase inhibitor by increasing the rate of loop insertion through hinge region mutations alone. We found that none of the three variants examined showed any detectable proteinase inhibitory properties. However, replacement of the P14 arginine residue of ovalbumin by serine, either alone or in combination with changes of P12-P10 to alanine, resulted in a large increase in the rate of loop insertion into beta-sheet A following cleavage at the P1-P1' bond by porcine pancreatic elastase (PPE), as shown by the spontaneous formation of a loop-inserted form upon cleavage that has increased the thermal stability. From the magnitude of the increase in stability of the cleaved, loop-inserted forms of the P14 ovalbumin variants, as well as the accessibility of the P1-P1'-cleaved reactive center loop to further proteolysis at P8-P7, we concluded that the reactive center loop can only partially insert into beta-sheet A and therefore that ovalbumin is also defective in the ability of beta-sheet A to expand to fully accommodate the whole of the reactive center loop. This defect, through its effect on the extent and/or rate of loop insertion, is likely to be a principal reason for ovalbumin not being a proteinase inhibitor.

Animals↗

PubMed1996

Role of arginine 132 and lysine 133 in heparin binding to and activation of antithrombin.

The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The Kd values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the Kd values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.

Antithrombin III↗

PubMed1996

Antithrombin-heparin affinity reduced by fucosylation of carbohydrate at asparagine 155.

The two human plasma antithrombin isoforms, alpha and beta, differ in glycosylation at asparagine 135. Only the alpha form carries carbohydrate at this position and has lower affinity for heparin than the beta form. We previously found additional heterogeneity in a recombinant N135Q antithrombin variant, evidenced by two isoforms with a 2-fold difference in heparin affinity [Turko, I. V., Fan, B., & Gettins, P. G. W. (1993) FEBS Lett. 335, 9-12]. To test whether this heterogeneity of heparin affinity results from specific glycosylation differences, we have determined the carbohydrate composition at the three remaining glycosylation sites, asparagine residues 96, 155, and 192, in each of the two N135Q isoforms, by a combination of peptide fragmentation and electrospray mass spectrometry. Patterns of glycosylation at residues 96 and 192 were similar for each isoform and showed the presence of mono-, bi-, and triantennary complex carbohydrate, as well as fucosylation of all types of chains. At position 155, however, there was a marked difference between the isoforms, with the form with lower heparin affinity being 97% fucosylated at this position, whereas the form with higher affinity for heparin was not fucosylated. Other differences in carbohydrate type showed no strong correlation between the two isoforms. We conclude that formation of the two heparin-affinity isoforms of N135Q antithrombin results from the specific difference in fucosylation at residue 155, which may result in different structural properties of the carbohydrate. Consistent with these findings was the elimination of heparin-affinity heterogeneity in a double N135Q-N155Q variant antithrombin. It is possible that fucosylation of antithrombin may occur in vivo as a means of modifying the physiological properties of the antithrombin through alteration of the amount of antithrombin bound to surface heparin-like species.

Amino Acid Sequence↗

PubMed1996

Mechanism of heparin activation of antithrombin. Evidence for reactive center loop preinsertion with expulsion upon heparin binding.

A heparin-induced conformational change is required to convert antithrombin from a slow to a fast inhibitor of factor Xa. It has been proposed [van Boeckel et al. (1994) Nat. Struct. Biol. 1, 423-425] that the reactive center residue P14 is inserted into beta-sheet A in native antithrombin and is displaced from the beta-sheet by heparin binding, thereby altering the conformation of the reactive center and making it a better target for factor Xa binding. To test this hypothesis, we have characterized a P14 serine --> tryptophan antithrombin variant. From changes in tryptophan fluorescence upon heparin binding, increased affinity for heparin, and partial activation of the variant against factor Xa, we conclude that the proposed mechanism of heparin activation is correct with respect to loop expulsion and that it may consequently be possible to create more highly activated antithrombin variants through suitable hinge region substitutions.

Antithrombin III↗