Research Article: Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase ScsC from Proteus mirabilis

Date Published: March 01, 2019

Publisher: International Union of Crystallography

Author(s): Emily J. Furlong, Fabian Kurth, Lakshmanane Premkumar, Andrew E. Whitten, Jennifer L. Martin.

http://doi.org/10.1107/S2059798319000081

Abstract

The structure and function of a bacterial trimeric protein disulfide isomerase was investigated by characterising two variants in which key structural regions were deleted. In one case the effect on structure and function was predictable, while in the other we found unintended consequences for both structure and function.

Partial Text

Protein disulfide isomerases are enzymes that proofread and shuffle incorrect disulfide bonds in misfolded protein substrates, and are important for the correct folding and function of many secreted proteins (Berkmen et al., 2005 ▸; Hiniker & Bardwell, 2004 ▸). The prototypical bacterial di­sulfide isomerase is disulfide-bond (Dsb) protein C from Escherichia coli (EcDsbC; Zapun et al., 1995 ▸; Missiakas et al., 1994 ▸; Shevchik et al., 1994 ▸). EcDsbC functions as a dimer; when the N-terminal dimerization domain is deleted the resulting protein lacks disulfide isomerase activity (Sun & Wang, 2000 ▸) and is also unable to interact with its redox partner EcDsbD (Goldstone et al., 2001 ▸). Each EcDsbC protomer has a thioredoxin-fold catalytic domain with a redox-active motif consisting of two cysteines separated by two other amino acids (CXXC; McCarthy et al., 2000 ▸). The disulfide-isomerase activity of EcDsbC requires that the catalytic cysteines are in the dithiol-reduced form (Darby et al., 1998 ▸), and this form is generated by the interaction of EcDsbC with its redox-partner membrane protein EcDsbD (Goldstone et al., 2001 ▸).

This study focused on characterizing two variants of PmScsC, which in its native form is a highly dynamic, trimeric disulfide isomerase. The native PmScsC protomer consists of an N-terminal trimerization stem with an 11-amino-acid flexible linker connected to a C-terminal thioredoxin-fold catalytic domain harbouring a CXXC active site. To better understand the role of the trimerization stem and flexible linker, we designed the variants PmScsCΔN and PmScsCΔLinker. PmScsCΔN lacks the first 41 amino acids of the native PmScsC construct; its loss of disulfide isomerase activity and gain of dithiol oxidase activity has been reported previously (Furlong et al., 2017 ▸). PmScsCΔLinker is newly reported here and lacks the 11 amino acids that form the flexible linker. Here, we have reported the structure of PmScsCΔN and analysed the structure and biochemical function of PmScsCΔLinker.

 

Source:

http://doi.org/10.1107/S2059798319000081

 

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