Role of the P6−P3‘ Region of the Serpin Reactive Loop in the Formation and Breakdown of the Inhibitory Complex

148

130

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6 -P3 proteinase binding sequence excluding P1-P1 to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2 residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6 -P3 residues flanking the primary P1-P1 recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6 -P3 region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.Antithrombin is a member of the serpin family of serine proteinase inhibitors whose primary physiologic function is to inhibit and thereby regulate several serine proteinases in the blood coagulation pathway (1, 2). Inhibition of these proteinases results from the serpin trapping the enzymes as stable acyl-intermediate complexes of a regular substrate reaction through a major conformational change. A unique feature of antithrombin is that it requires activation by the polysaccharide, heparin, to inhibit its main target enzymes, thrombin and factor Xa, at a physiologically significant rate. Heparin activates antithrombin through two distinct mechanisms whose relative contribution depends on the proteinase inhibited (3, 4). In both mechanisms, antithrombin binds to a sequence-specific pentasaccharide in heparin (5) which induces an activating conformational change in the reactive center loop of the serpin (6 -9). Such conformational changes are alone sufficient to accelerate antithrombin inactivation of factor Xa through an allosteric mechanism. By contrast, the conformational changes negligibly affect the rate of thrombin inhibition, heparin a...

Section: Discussionmentioning

confidence: 99%

Heparin Enhances the Specificity of Antithrombin for Thrombin and Factor Xa Independent of the Reactive Center Loop Sequence

Chuang

Swanson

Raja

et al. 2001

148

130

“…Their concentration and activ-ity were determined as described previously (20,21). The stoichiometry of inhibition against chymotrypsin was found to be 1, indicating full activity.…”

Section: Methodsmentioning

confidence: 99%

Conformational Change and Intermediates in the Unfolding of α1-Antichymotrypsin

Pearce¹,

Rubin²,

Bottomley³

2000

Self Cite

Serpins are the prototypical members of the conformational disease family, a group of proteins that undergoes a change in shape that subsequently leads to tissue deposition. One specific example is ␣ 1 -antichymotrypsin (ACT), which undergoes misfolding and aggregation that has been implicated in emphysema and Alzheimer's disease. In this study we have used guanidine hydrochloride (GdnHCl)-induced denaturation to investigate the conformational changes involved in the folding and unfolding of ACT. When the reaction was followed by circular dichroism spectroscopy, one stable intermediate was observed in 1.5 M GdnHCl. The same experiment monitored by fluorescence revealed a second intermediate formed in 2.5 M GdnHCl. Both these intermediates bound the hydrophobic dye ANS. These data suggest a four-state model for ACT folding N 7 I 1 7 I 2 7 U. I 1 and I 2 both have a similar loss of secondary structure (20%) compared with the native state. In I 2 , however, there is a significant loss of tertiary interactions as revealed by changes in fluorescence emission maximum and intensity. Kinetic analysis of the unfolding reaction indicated that the native state is unstable with a fast rate of unfolding in water of 0.4 s ؊1 . The implications of these data for both ACT function and associated diseases are discussed.Conformational disorders, as defined by Carrell and colleagues (1, 2), arise when a protein undergoes a change in shape that leads to self-association, tissue deposition, and disease. The serpin superfamily includes some of the most studied proteins involved in this group of disorders. The misfolding and aggregation of serpins have been implicated in diseases ranging from emphysema and liver disease, to cancer and dementia (3-5). It is, however, extremely surprising that so little is known about the basic folding pathways of this class of protein.A number of studies have shown that ␣ 1 -antitrypsin unfolds through one intermediate (6 -8), and recent studies with maspin (9) and plasminogen activator inhibitor-1 (10) have shown similar results.The folding pathway of ␣ 1 -antichymotrypsin (ACT), 1 which is involved in regulation of several proteinases, including chymotrypsin, cathepsin G, and mast cell chymase, has, so far, received no attention. ACT is an acute phase DNA binding glycoprotein that possesses the characteristic serpin tertiary fold, composed of three ␤-sheets (A, B, and C) surrounded by nine ␣-helices (A-I) (11, 12). X-ray crystallographic and biochemical data have shown that ACT is capable of adopting several different conformations (12, 13). Cleavage of the reactive center loop (RCL) by noncognate proteinases causes full insertion of the RCL into the A ␤-sheet, forming an additional ␤-strand, which confers additional stability to the protein (12). The recent structure of an ACT variant demonstrated that partial insertion of the RCL was also possible, concomitant with unwinding and insertion of part of the F-helix, which lies on the A ␤-sheet (14). RCL sheet insertion is also possible in the absence o...

“…dissociating after hours or days into active enzyme and cleaved, inactive inhibitor. The identity of the P 1 residue is the major determinant of serpin inhibitory specificity, but other RCL residues, as well as serpin body-proteinase body interactions, are also involved (18,19). Thus empirical testing and kinetic studies are required to identify target proteinases.…”

Section: /Ebi Data Bank With Accession Number(s)z49890 (Wsz1a) Y1148mentioning

confidence: 99%

Inhibitory Serpins from Wheat Grain with Reactive Centers Resembling Glutamine-rich Repeats of Prolamin Storage Proteins

Østergaard¹,

Rasmussen²,

Roberts³

et al. 2000

114

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P 1 -P 1 Gln-Gln, and WSZ2b at P 2 -P 1 LeuArg-one of two overlapping reactive centers. WSZ1c with P 1 -P 1 Leu-Gln was the fastest inhibitor of chymotrypsin (k a ‫؍‬ 1 The serpins constitute a superfamily of versatile proteins participating in the regulation of complex proteolytic systems (1, 2). Most serpins are serine proteinase inhibitors of chymotrypsin-like enzymes, but a few have been found to inhibit cysteine proteinases (3), and some are non-inhibitory. Many functionally distinct serpins have been identified in higher eukaryotes, some in viruses, but none in yeast or bacteria (4, 5).Mammalian serpins have been shown to participate in a growing number of extra-and intracellular physiological processes, including blood coagulation, complement activation, remodeling of the extracellular matrix, and hormone transport.The only plant serpin characterized in detail with respect to inhibitory specificity is recombinant barley (Hordeum vulgare) serpin rBSZx 1 (6 -8), but BSZx (GenBank™ accession number X97636) has not been detected in the plant. In addition, two serpins from barley grain, BSZ4 (7, 9 -11) and BSZ7 (GenBank™ accession number CAA64599) (12, 13), and one from wheat (Triticum aestivum) grain (7, 14, 15) have been studied. Despite the high concentration of these inhibitors in the grains of monocot cereals (up to 4% total protein), the physiological functions of plant serpins remain unknown. Increasing numbers of putative serpin genes and serpin mRNAs have been identified from other monocot plants, including rice and wild oats, from eudicot plants, including tomato, cotton, and the model plant Arabidopsis thaliana, and from the non-vascular plant Physcomitrella patens (EMBL/GenBank), suggesting that serpins are widespread in the plant kingdom.Inhibitory serpins are metastable proteins (Ͼ40 kDa) employing a unique "suicide substrate" mechanism of irreversible inhibition very different from the reversible "standard mechanism" used by other proteinase inhibitors (Ͻ25 kDa) (1). The serpin in its native, active conformation has a flexible reactive center loop (RCL) located near the C terminus and protruding from the main body of the protein...