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1.
Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1. 相似文献
2.
Nukuna BN Penn MS Anderson VE Hazen SL 《The Journal of biological chemistry》2004,279(48):50132-50141
Plasminogen activator inhibitor type 1 (PAI-1) plays key regulatory roles in fibrinolysis, cell migration, and tissue remodeling. A regulatory protein without known catalytic activity, PAI-1 modulates plasminogen activators through protein-protein interactions. Although global conformational alterations that occur in PAI-1 determine its regulatory activity, comprehensive assessments of concurrent dynamic, structural, and functional alterations of this critical regulatory protein have not yet been clearly defined. X-ray crystallographic studies have described four distinct PAI-1 conformational states: active, latent, reactive center loop peptide-annealed (RCL-PA), and cleaved mutant. In this study, backbone amide hydrogen-deuterium exchange detected by mass spectrometry was used to characterize dynamic and structural alterations of human PAI-1 (hPAI-1) in relation to its function. hPAI-1 conformers were defined by surface mapping the solvent-accessible sites for strategic secondary structural components of the protein. We observed a global protection from solvent for a majority of peptides in the latent conformer relative to the active conformer. Significant differences were observed in the RCL, helix A, helix D, and sheet 1C, and these regions were markedly more dynamic or solvent-exposed in the active conformation. The RCL-PA form adopts an intermediate conformational state between the active and the latent conformers. Our results demonstrate that the most dynamic regions of PAI-1 (the RCL, helices D and A, and sheet 5A) are flexible in the transition toward latency. They also show that the dynamic surface structures of the active, latent, and peptide-annealed conformers of PAI-1 are underestimated by theoretical solvent accessibility calculations derived from crystallographic data. 相似文献
3.
P J Declerck M De Mol D E Vaughan D Collen 《The Journal of biological chemistry》1992,267(17):11693-11696
Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity. 相似文献
4.
The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted 总被引:3,自引:0,他引:3
Hägglöf P Bergström F Wilczynska M Johansson LB Ny T 《Journal of molecular biology》2004,335(3):823-832
Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1' residues acting as a "bait" for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into beta-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor-donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in beta-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into beta-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form. 相似文献
5.
Dupont DM Blouse GE Hansen M Mathiasen L Kjelgaard S Jensen JK Christensen A Gils A Declerck PJ Andreasen PA Wind T 《The Journal of biological chemistry》2006,281(47):36071-36081
Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule. 相似文献
6.
Lawrence DA Olson ST Muhammad S Day DE Kvassman JO Ginsburg D Shore JD 《The Journal of biological chemistry》2000,275(8):5839-5844
The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing beta-sheet, serpin beta-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P(14) hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation. 相似文献
7.
Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of plasminogen activators (uPA and tPA) and thus plays a central role in fibrinolysis. The spontaneous insertion of its reactive centre loop (RCL) into β-sheet A is responsible for its irreversible conversion into the inactive latent form. In this study, we used two peptides mimicking residues P14-P9 and P8-P3 of the RCL so as to understand this dynamic process. We show that both peptides inhibit the formation of PAI-1/uPA and PAI-1/tPA complexes via two different mechanisms. Targeting the N-terminal part of the loop induces the cleavage of PAI-1 by the proteases uPA/tPA while targeting its C-terminal part greatly favors the irreversible formation of latent PAI-1. 相似文献
8.
I Verhamme J O Kvassman D Day S Debrock N Vleugels P J Declerck J D Shore 《The Journal of biological chemistry》1999,274(25):17511-17517
The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its beta-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA).PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probe N, N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9.NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3-1.8 x 10(4) M-1 s-1), resulting in a 4000-fold faster inactivation at 4.2 microM antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody. 相似文献
9.
Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single-chain human tPA. 总被引:7,自引:2,他引:5
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M Renatus R A Engh M T Stubbs R Huber S Fischer U Kohnert W Bode 《The EMBO journal》1997,16(16):4797-4805
Tissue type plasminogen activator (tPA) is the physiological initiator of fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then degrades fibrin with relatively broad specificity. Unlike other chymotrypsin family serine proteinases, tPA is proteolytically active in a single-chain form. This form is also preferred for therapeutic administration of tPA in cases of acute myocardial infarction. The proteolytic cleavage which activates most other chymotrypsin family serine proteinases increases the catalytic efficiency of tPA only 5- to 10-fold. The X-ray crystal structure of the catalytic domain of recombinant human single-chain tPA shows that Lys156 forms a salt bridge with Asp194, promoting an active conformation in the single-chain form. Comparisons with the structures of other serine proteinases that also possess Lys156, such as trypsin, factor Xa and human urokinase plasminogen activator (uPA), identify a set of secondary interactions which are required for Lys156 to fulfil this activating role. These findings help explain the anomalous single-chain activity of tPA and may suggest strategies for design of new therapeutic plasminogen activators. 相似文献
10.
Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central beta-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to alpha-helix F (alphaHF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k(lim)) of RCL insertion for reactions with beta-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k(lim) for tPA but decreased the k(lim) for beta-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of alphaHF, decreased k(lim) and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for beta-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k(lim) but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to alphaHF, controls bifurcation between the inhibitory and the substrate pathways. 相似文献
11.
Plasminogen activator inhibitor-1 (PAI-1) acts as the major inhibitor of fibrinolysis by inhibiting tissue-type and urokinase-type plasminogen activators. Although it shares a common tertiary structure with other serine protease inhibitors, PAI-1 is unique in its conformational lability, which allows conversion of the active form to the latent conformation under physiological conditions. Therefore, recombinant PAI-1 expressed in eukaryotic or prokaryotic cells almost always contains its inactive, latent form, with very low specific activity. In this study, we developed a simple and efficient method for purifying the active form of recombinant PAI-1 rather than the latent conformation from PAI-1 overexpressing Escherichia coli cells. The overall level of expression and the amount of PAI-1 found in inclusion bodies were found to increase with culture temperature and with time after induction. Refolding of unfolded PAI-1 from inclusion bodies and ion-exchange column chromatography were sufficient to purify PAI-1. The purified protein yielded a single, 43kDa protein band upon SDS-polyacrylamide gel electrophoresis, and it efficiently inhibited tissue-type and urokinase-type plasminogen activators similar to PAI-1 from natural sources. Activity measurements showed that PAI-1 purified from inclusion bodies exhibited a specific activity near the theoretical maximum, unlike PAI-1 prepared from cytosolic fractions. Conformational analysis by urea gel electrophoresis also indicated that the PAI-1 protein purified from inclusion bodies was indeed in its active conformation. 相似文献
12.
The serpins are of general protein chemical interest due to their ability to undergo a large conformational change consisting of the insertion of the reactive centre loop (RCL), which becomes strand 4, into the central beta sheet A. To make space for the incoming RCL, the 'shutter region' opens by the beta strands 3A and 5A sliding apart over the underlying alpha helix B. Loop insertion occurs during the formation of complexes of serpins with their target serine proteinases and during latency transition. This type of loop insertion is unique to plasminogen activator inhibitor-1 (PAI-1). We report here that amino-acid substitutions in a buried cluster of three residues forming a hydrogen bonding network in the shutter region drastically accelerate PAI-1 latency transition; that the rate was in all cases normalized by the PAI-1 binding protein vitronectin; and that substitution of an adjacent beta strand 5A Lys residue, believed to anchor beta strand 5A to other secondary structural elements, had differential effects on the rates of latency transition in the absence and the presence of vitronectin, respectively. An overlapping, but not identical set of substitutions resulted in an increased tendency to substrate behaviour of PAI-1 at reaction with its target proteinases. These findings show that vitronectin regulates the movements of the RCL through conformational changes of the shutter region and beta strand 5A, are in agreement with RCL insertion proceeding by different routes during latency transition and complex formation, and contribute to the biochemical basis for the potential use of PAI-1 as a therapeutic target in cancer and cardiovascular diseases. 相似文献
13.
The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation. 相似文献
14.
Julie S B?dker Troels Wind Jan K Jensen Martin Hansen Katrine E Pedersen Peter A Andreasen 《European journal of biochemistry》2003,270(8):1672-1679
Plasminogen activator inhibitor-1 (PAI-1) belongs to the serpin family of serine proteinase inhibitors. Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P1 residue of the reactive centre loop (RCL) of the serpin, followed by insertion of the RCL into beta-sheet A of the serpin. Concomitantly, there are conformational changes in the flexible joint region lateral to beta-sheet A. We have now, by site-directed mutagenesis, mapped the epitope for a monoclonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acting on beta-sheet A and the flexible joint region. Curiously, the epitope is localized in alpha-helix C and the loop connecting alpha-helix I and beta-strand 5A, on the side of PAI-1 opposite to beta-sheet A and distantly from the flexible joint region. By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody-induced conformational change from the epitope to the rest of the molecule. We have thus provided evidence for communication between secondary structural elements not previously known to interact in serpins. 相似文献
15.
Troels Wind Jan K Jensen Daniel M Dupont Paulina Kulig Peter A Andreasen 《European journal of biochemistry》2003,270(8):1680-1688
The serpin plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis. PAI-1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop (RCL) as strand 4 in beta-sheet A, thereby converting to the so-called latent state. We have investigated the importance of the amino acid sequence of alpha-helix F (hF) and the connecting loop to s3A (hF/s3A-loop) for the rate of latency transition. We grafted regions of the hF/s3A-loop from antithrombin III and alpha1-protease inhibitor onto PAI-1, creating eight variants, and found that one of these reversions towards the serpin consensus decreased the rate of latency transition. We prepared 28 PAI-1 variants with individual residues in hF and beta-sheet A replaced by an alanine. We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case. Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complex formation with target proteases. The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop-sheet interactions in serpins. 相似文献
16.
Komissarov AA Andreasen PA Bødker JS Declerck PJ Anagli JY Shore JD 《The Journal of biological chemistry》2005,280(2):1482-1489
The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo. 相似文献
17.
T Kirkegaard S Jensen S L Schousboe H H Petersen R Egelund P A Andreasen K W Rodenburg 《European journal of biochemistry》1999,263(2):577-586
The serpin (serine proteinase inhibitor) family is of general protein chemical interest because of its ability to undergo large conformational changes, in which the surface-exposed reactive centre loop (RCL) is inserted as strand 4 in the large central beta-sheet A. Loop insertion is an integral part of the inhibitory mechanism and also takes place at conversion of serpins to the latent state, occurring spontaneously only in plasminogen activator inhibitor-1 (PAI-1). We have investigated the importance of beta-strand 5A residues for the activity and latency transition of PAI-1. An approximately fourfold increase in the rate of latency transition resulted from His-substitution of Gln324 (position 334 in the alpha(1)-proteinase inhibitor template numbering), which interacts with the underlying alpha-helix B. The side chains of Gln321 and Lys325 (template residues 331 and 335, respectively) form hydrogen bonds to the peptide backbone of a loop connecting alpha-helix F and beta-strand 3A. While substitution with Ala of Glu321 had only minor effects on the properties of PAI-1, substitution with Ala of Lys325 led to stabilization of the inhibitory activity at incubation conditions leading to conversion of wild-type PAI-1 to a substrate form, and to an anomalous reaction towards a monoclonal antibody, which induced a delay in the latency transition of the mutant, but not wild-type PAI-1. We conclude that the anchoring of beta-strand 5A plays a crucial role in loop insertion. These findings provide new information about the mechanism of an important example of protein conformational changes. 相似文献
18.
19.
Single fluorescence probes along the reactive center loop reveal site‐specific changes during the latency transition of PAI‐1
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Tihami Qureshi Cynthia B. Peterson 《Protein science : a publication of the Protein Society》2016,25(2):487-498
The serine protease inhibitor (serpin), plasminogen activator inhibitor‐1 (PAI‐1), is an important biomarker for cardiovascular disease and many cancers. It is therefore a desirable target for pharmaceutical intervention. However, to date, no PAI‐1 inhibitor has successfully reached clinical trial, indicating the necessity to learn more about the mechanics of the serpin. Although its kinetics of inhibition have been extensively studied, less is known about the latency transition of PAI‐1, in which the solvent‐exposed reactive center loop (RCL) inserts into its central β‐sheet, rendering the inhibitor inactive. This spontaneous transition is concomitant with a large translocation of the RCL, but no change in covalent structure. Here, we conjugated the fluorescent probe, NBD, to single positions along the RCL (P13‐P5′) to detect changes in solvent exposure that occur during the latency transition. The results support a mousetrap‐like RCL‐insertion that occurs with a half‐life of 1–2 h in accordance with previous reports. Importantly, this study exposes unique transitions during latency that occur with a half‐life of ~5 and 25 min at the P5′ and P8 RCL positions, respectively. We hypothesize that the process detected at P5′ represents s1C detachment, while that at P8 results from a steric barrier to RCL insertion. Together, these findings provide new insights by characterizing multiple steps in the latency transition. 相似文献
20.
Bernd R. Gardill Michael R. Vogl Hai-Yan Lin Geoffrey L. Hammond Yves A. Muller 《PloS one》2012,7(12)
Corticosteroid-binding globulin (CBG) transports glucocorticoids and progesterone in the blood and thereby modulates the tissue availability of these hormones. As a member of the serine protease inhibitor (SERPIN) family, CBG displays a reactive center loop (RCL) that is targeted by proteinases. Cleavage of the RCL is thought to trigger a SERPIN-typical stressed-to-relaxed (S-to-R) transition that leads to marked structural rearrangements and a reduced steroid-binding affinity. To characterize structure-function relationships in CBG we studied various conformational states of E. coli-produced rat and human CBG. In the 2.5 Å crystal structure of human CBG in complex with progesterone, the RCL is cleaved at a novel site that differs from the known human neutrophil elastase recognition site. Although the cleaved RCL segment is five residues longer than anticipated, it becomes an integral part of β-sheet A as a result of the S-to-R transition. The atomic interactions observed between progesterone and CBG explain the lower affinity of progesterone in comparison to corticosteroids. Surprisingly, CD measurements in combination with thermal unfolding experiments show that rat CBG fails to undergo an S-to-R transition upon proteolytic cleavage of the RCL hinting that the S-to-R transition observed in human CBG is not a prerequisite for CBG function in rat. This observation cautions against drawing general conclusions about molecular mechanisms by comparing and merging structural data from different species. 相似文献