Inhibitory mechanism of serpins. Mobility of the C-terminal region of the reactive-site loop.
The reactive-site loops of serpins are characterized by a defined mobility where the loop adopts a new secondary structure as an essential part of the inhibitory process. While the importance of mobility in the N-terminal region of the reactive-site loop has been well studied, the role of mobility in the C-terminal portion has not been investigated. The requirements for mobility of the C-terminal portion of the reactive-site loop of alpha1-antitrypsin were investigated by creating a disulfide bridge between the P'3 residue and residue 283 near the top of strand 2C; this disulfide would restrict the mobility of the C-terminal portion of the reactive-site loop by locking together strands 1 and 2 of the C beta-sheet. The engineered disulfide bond had no effect on the inhibitory activity of alpha1-antitrypsin, indicating that there is no requirement for mobility in this region of the molecule. Moreover, these results, coupled with those from molecular modeling, indicate that insertion into the A beta-sheet of the intact reactive-loop beyond P12 is not rate-limiting for the formation of the stable complex. The engineered disulfide bond should also prove useful in the creation of more stable serpin variants; for example, such a bond in plasminogen activator inhibitor-1 would prevent it from becoming latent by locking strand 1C onto the C beta-sheet.
Hopkins
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PC
Department of Haematology, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom.
Chang
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WS
Wardell
M R
MR
Stone
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SR
D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*
D004220:Disulfides; D000515:alpha 1-Antitrypsin; D019272:Leukocyte Elastase