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
Horst Joachim SchirraD J Craik

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 K(i) of <5 nM, C4A/C41A-T1 has a weaker K(i) of approximately 350 nM, and the potency of the C8A/C37A variant is further decreased to a K(i) of approximately 1.8 microM. 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 st...Continue Reading

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Citations

Nov 17, 2012·BMC Plant Biology·Manasi MishraAshok P Giri
Mar 7, 2020·Nature Communications·Alexandra RehnJohannes Buchner
Jun 11, 2021·BMC Plant Biology·Nikhilesh K YadavAshok P Giri

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