Evidence for the extent of insertion of the active site loop of intact .alpha.1 proteinase inhibitor in .beta.-sheet A

Schulze, Andreas; Frohnert, Paul W.; Engh, Richard A.; Huber, Robert

doi:10.1021/bi00148a017

Cited by 78 publications

(50 citation statements)

References 18 publications

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“…It has also been shown for cc,-proteinase inhibitor that uptake of exogenous free peptides of varying length, with sequences of the a,-proteinase inhibitor s4A strand, affect the inhibitor activity of the serpin [21]. Peptides of length and sequence corresponding to Pl-P12, Pl-P14, P8-P14 and P4P14 all abolish the inhibitor activity of a,-proteinase inhibitor and transform it into a trypsin substrate.…”

Section: Resultsmentioning

confidence: 99%

“…The peptide insertion studies of Schulze et al [21] also imply that failure of the P12-P14 segment s4A to insert is sufficient to abolish inhibitor activity. However, the results on antithrombinHamilton reported here do not support the converse proposition that s4A insertion in P12-P14 is sufficient to confer inhibitor activity on a serpin.…”

Section: Ht Wright Ma Blajchmanlfebsmentioning

confidence: 97%

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Proteolytically cleaved mutant antithrombin‐Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins

Wright

Blajcliman

1994

FEBS Letters

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The serpin family of proteins consists primarily of proteinase inhibitors which form tight complexes with target proteinases. Inhibitor serpins are cleaved by proteinase and undergo a large conformational change in which the polypeptide segment terminating at the target reactive site, at which cleavage takes place, inserts itself as an additional strand, s4A, in the center of a preexisting/?-sheet. This change in conformation increases the stability towards denaturation of the cleaved serpin relative to the native uncleaved form. Mutant serpins with single amino acid changes in the s4A strand have been identified, and in most cases these are proteinase substrates but not inhibitors. We have measured the stability to denaturation of one of these non-inhibitor substrate mutants, antithrombin-Hamilton, which has an Ala + Thr change at position P12 in strand s4A. We find that it undergoes the transformation to the more stable form which is observed for inhibitor serpins, from which we conclude that the Ala + Thr change in antithrombin-Hamilton does not prevent insertion of s4A into B-sheet A in the cleaved form.

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Section: Resultsmentioning

confidence: 99%

Section: Ht Wright Ma Blajchmanlfebsmentioning

confidence: 97%

Proteolytically cleaved mutant antithrombin‐Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins

Wright

Blajcliman

1994

FEBS Letters

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“…The nature of this rearrangement was suggested by Lobermann et al, but was first observed in part in the X-ray structure of proteolytically modified ovalbumin, PLAK [5]. The structure of intact ovalbumin followed [6], showing an a-helical structure for the strand corresponding to the binding site of serpin inhibitors, similar [14], although these structures also leave unanswered many questions regarding the serpin inhibitory mechanism.…”

Section: X-ray Structuresmentioning

confidence: 96%

Structural aspects of serpin inhibition

et al. 1994

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The essential roles of proteins of the serpin family in many physiological processes, along with new discoveries of their unique folding properties, have attracted intense interest in recent years. Many serpins display unusual mobile behavior attributed to rearrangements of a-helical or /J-sheet domains, whereby large scale transitions accompany a variety of functions, including inactivation. This unusual behavior was first recognized with the X-ray structure of modified al-proteinase inhibitor. Subsequent experiments, including new X-ray structures, have revealed a surprising variety of conformations which are functionally important but only partially understood. We review here experimental evidence for conformations relevant to the serpin inhibitory mechanism.

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“…Proteinase inhibitor function depends critically on mobility of the reactive loop, and in particular on its ability to stably insert into central ␤-sheet A during complex formation with target enzymes. Serpins in complex with their target proteinases are inferred to have their reactive loops partially inserted into their A-sheets as strand 4A (s4A) (3)(4)(5)(6). This partially inserted conformation is intermediate between those of native serpins, which have 5-stranded A-sheets (7,8), and latent and cleaved serpins which have fully 6-stranded A-sheets (9 -13).…”

mentioning

confidence: 99%

Identification of the Antithrombin III Heparin Binding Site

Ersdal-Badju

Zuo

et al. 1997

Journal of Biological Chemistry

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The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring ␤-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Bjö rk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-Å long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central ␤-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

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Evidence for the extent of insertion of the active site loop of intact .alpha.1 proteinase inhibitor in .beta.-sheet A

Cited by 78 publications

References 18 publications

Proteolytically cleaved mutant antithrombin‐Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins

Proteolytically cleaved mutant antithrombin‐Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins

Structural aspects of serpin inhibition

Identification of the Antithrombin III Heparin Binding Site

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