Selective removal of individual disulfide bonds within a potato type II serine proteinase inhibitor from Nicotiana alata reveals differential stabilization of the reactive-site loop

Schirra, Horst Joachim, Guarino, Rosemary F., Anderson, Marilyn A. and Craik, David J. (2009) Selective removal of individual disulfide bonds within a potato type II serine proteinase inhibitor from Nicotiana alata reveals differential stabilization of the reactive-site loop. Journal of Molecular Biology, 395 3: 609-626. doi:10.1016/j.jmb.2009.11.031


Author Schirra, Horst Joachim
Guarino, Rosemary F.
Anderson, Marilyn A.
Craik, David J.
Title Selective removal of individual disulfide bonds within a potato type II serine proteinase inhibitor from Nicotiana alata reveals differential stabilization of the reactive-site loop
Formatted title
Selective removal of individual disulfide bonds within a potato type II serine proteinase inhibitor from Nicotiana alata reveals differential stabilization of the reactive-site loop
Journal name Journal of Molecular Biology   Check publisher's open access policy
ISSN 0022-2836
1089-8638
Publication date 2009-11-17
Sub-type Article (original research)
DOI 10.1016/j.jmb.2009.11.031
Volume 395
Issue 3
Start page 609
End page 626
Total pages 18
Editor Peter Wright
Jonathan Karn
Place of publication United Kingdom
Publisher Academic Press
Collection year 2010
Language eng
Subject C1
060112 Structural Biology (incl. Macromolecular Modelling)
820301 Cotton
960413 Control of Plant Pests, Diseases and Exotic Species in Farmland, Arable Cropland and Permanent Cropland Environments
Abstract The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4–C41 and C8–C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a Ki of < 5 nM, C4A/C41A-T1 has a weaker Ki of not, vert, similar 350 nM, and the potency of the C8A/C37A variant is further decreased to a Ki of not, vert, similar 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4–C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond–nanosecond) timescale. In contrast, no comparable interactions are present around the C8–C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8–C37 and C4–C41 disulfide bonds. The C8–C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4–C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond–nanosecond timescale.
Keyword proteinase inhibitor
potato type II family
NMR spectroscopy
protein structure
structure-function relationship
Q-Index Code C1
Q-Index Status Confirmed Code

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
School of Chemistry and Molecular Biosciences
 
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Created: Sun, 21 Feb 2010, 00:03:53 EST