Intrinsic specificity of the reactive site loop of alpha1-antitrypsin, alpha1-antichymotrypsin, antithrombin III, and protease nexin I.
Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.
Djie
M Z
MZ
Department of Haematology, University of Cambridge, MRC Centre, Hills Road, Cambridge, CB2 2QH, United Kingdom.
Stone
S R
SR
Le Bonniec
B F
BF
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor; D000990:Antithrombin III / Q000737:chemistry* / Q000502:physiology; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D058258:Serpin E2; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry* / Q000502:physiology; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000502:physiology
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D015842:Serine Proteinase Inhibitors; D058258:Serpin E2; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D000990:Antithrombin III