Serpins: the uncut version

Goldsmith, E.J.; Mottonen, James M.

doi:10.1016/s0969-2126(00)00025-3

Cited by 21 publications

(10 citation statements)

References 18 publications

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“…In the search for a candidate among the 13 histidine residues of human PAI-1, we studied the structure of the latent inhibitor and focused on histidine residues that could have a direct effect on binding of peptides to sheet A. This approach is justified by the fact that apart from the reactive center loop, the differences between serpins with 5 or 6 strands in sheet A are predominantly observed in this sheet (33)(34)(35) and its immediate vicinity. More profound changes may accompany formation of latent PAI-1 (6) but are not likely to be required for insertion of the octamer peptides.…”

Section: Discussionmentioning

confidence: 99%

“…In the latent inhibitor the presumably neutral His-143 (the crystals were grown at pH 8.2 (36)) is seen at hydrogen bond distance to the ␥-oxygen of Thr-142 and the carbonyl oxygen of Trp-139, both in helix F. In the active inhibitor, a positive charge on the histidine in addition to structural differences may allow the imidazolium ring to rotate into a different position, where it may stabilize helix F by neutralizing its dipole field or by forming a hydrogen bond to sheet A. Although there is no suitable acceptor within distance for the latter bond in latent PAI-1, it can be seen from the differences between latent PAI-1 and nonrelaxed serpins (35) that sheet A in the former has expanded in the region beneath helix F. In the active inhibitor the side chain oxygen of Thr-94 in strand 2A and one of the nitrogens of His-143 may very well be within hydrogen bond distance. The spatial relationships pertinent to this discussion are illustrated by the stereodiagram of the ␣-carbon backbone SCHEME 2.…”

Section: Discussionmentioning

confidence: 99%

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The Acid Stabilization of Plasminogen Activator Inhibitor-1 Depends on Protonation of a Single Group That Affects Loop Insertion into β-Sheet A

Kvassman

Lawrence

Shore

1995

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor-1 (PAI-1) spontaneously adopts an inactive or latent conformation by inserting the N-terminal part of the reactive center loop as strand 4 into the major ␤-sheet (sheet A). To examine factors that may regulate reactive loop insertion in PAI-1, we determined the inactivation rate of the inhibitor in the pH range 4.5-13. Below pH 9, inactivation led primarily to latent PAI-1, and one predominant effect of pH on the corresponding rate constant could be observed. Protonation of a group exhibiting a pK a of 7.6 (25°C, ionic strength ‫؍‬ 0.15 M) reduced the rate of formation of latent PAI-1 by a factor of 35, from 0.17 h ؊1 at pH 9 to about 0.005 h ؊1 below pH 6. The ionization with a pK a 7.6 was found to have no effect on the rate by which PAI-1 inhibits trypsin and is therefore unlikely to change the flexibility of the loop or the orientation of the reactive center. The peptides Ac-TEAS-SSTA and Ac-TVASSSTA (cf. P14-P7 in the reactive loop of PAI-1) formed stable complexes with PAI-1 and converted the inhibitor to a substrate for tissue type plasminogen activator. We found that peptide binding and formation of latent PAI-1 are mutually exclusive events, similarly affected by the pK a 7.6 ionization. This is direct evidence that external peptides can substitute for strand 4 in ␤-sheet A of PAI-1 and that the pK a 7.6 ionization regulates insertion of complementary, internal or external, strands into this position. A model that accounts for the observed pH effects is presented, and the identity of the ionizing group is discussed based on the structure of latent PAI-1. The group is tentatively identified as His-143 in helix F, located on top of sheet A.Plasminogen activator inhibitor, PAI-1, is a member of the serpin family of serine proteinase inhibitors and inhibits both tPA 1 and urokinase plasminogen activator (1, 2) as well as trypsin (3). The serpins (4), which include most of the inhibitors that regulate blood coagulation and fibrinolysis, are structurally homogenous and are distinguished functionally from other types of protein proteinase inhibitors primarily by the ability to form SDS-stable complexes with target proteinases. The nature of these complexes and the mechanism by which they are formed are poorly understood.Active serpins are metastable folding intermediates with considerable conformational strain (5). They can relax by inserting the N-terminal portion of the reactive center loop as strand 4 in the major ␤-sheet that faces one side of the protein (sheet A) and thereby adopt an inactive, or latent, conformation. This process occurs spontaneously in PAI-1 (6) and has been induced in antithrombin III (5) and ␣ 1 -proteinase inhibitor (7). Inhibitory serpins attain an even greater stability after cleavage of the reactive loop, near or at the susceptible bond, and insertion of the N-terminal portion into ␤-sheet A (8 -10) or after forming complexes with peptides that mimic this part of the loop (11, 12). The fact that serpins with a completed sixstranded sheet...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Acid Stabilization of Plasminogen Activator Inhibitor-1 Depends on Protonation of a Single Group That Affects Loop Insertion into β-Sheet A

Kvassman

Lawrence

Shore

1995

Journal of Biological Chemistry

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“…Thus, kinetic control may be expected to occur in situations in which irreversibility is advantageous. The plasminogen activator inhibitor (PAI-1), a member of the serpin family of protease inhibitors, provides an interesting case in point [10]. After synthesis in vivo or refolding after denaturant treatment in vitro, PAI-1 folds first to a state which is an active protease inhibitor.…”

Section: Kinetic Versus Thermodynamic Controlmentioning

confidence: 99%

Influenza hemagglutinin: kinetic control of protein function

Baker

Agard

1994

Structure

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“…PAI-1 is a member of the serine protease inhibitor ("serpin") family of proteins (3)(4)(5)(6). The three-dimensional structure of a number of these proteins, either in an active or an inactive form, has been established (reviewed in (7)). The spatial structure of these proteins is similar, consistent with the view that their mechanism of action is governed by a common principle.…”

Section: Introductionmentioning

confidence: 99%

The Composition of Complexes between Plasminogen Activator Inhibitor 1, Vitronectin and either Thrombin or Tissue-type Plasminogen Activator

et al. 1997

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SummaryVitronectin (VN) is an obligatory cofactor for the inhibition of thrombin by plasminogen activator inhibitor 1 (PAI-1). It accelerates the rate of association between thrombin and PAI-1 more than two orders of magnitude. In contrast, VN does not accelerate the association between tissue-type plasminogen activator (t-PA) and PAI-1. Previously, we reported that the anti-PAI-1 monoclonal antibody (MoAb) CLB-2C8 binds to a short stretch of amino acids of PAI-1, located between residues 128 and 145, and prevents PAI-1 binding to VN. Furthermore, MoAb CLB-2C8 fully blocks the inhibitory activity of PAI-1 towards t-PA, emphasizing the importance of this area for the interaction with t-PA. Here, we show that this area is also required for the interaction between thrombin and PAI-1, since MoAb CLB-2C8 fully prevents inhibition of thrombin by PAI-1. In spite of similar structural requirements for the interaction between t-PA, PAI-1 and VN and between thrombin, PAI-1 and VN, the intermediate reaction products are clearly distinct. By employing surface plasmon resonance (SPR), using the BIAcore equipment, and by immunoprecipitation we demonstrate that, in the presence of VN, t-PA and PAI-1 form exclusively equimolar binary t-PA/PAI-1 complexes. Thrombin, PAI-1 and VN generate equimolar, binary thrombin/PAI-1 complexes and in addition equimolar, ternary complexes and multimers.

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Serpins: the uncut version

Abstract: The structure of active antithrombin, the first active serpin to be solved, sheds new light on the conformational forms of this important class of inhibitor.

Cited by 21 publications

References 18 publications

The Acid Stabilization of Plasminogen Activator Inhibitor-1 Depends on Protonation of a Single Group That Affects Loop Insertion into β-Sheet A

The Acid Stabilization of Plasminogen Activator Inhibitor-1 Depends on Protonation of a Single Group That Affects Loop Insertion into β-Sheet A

Influenza hemagglutinin: kinetic control of protein function

The Composition of Complexes between Plasminogen Activator Inhibitor 1, Vitronectin and either Thrombin or Tissue-type Plasminogen Activator

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