Abstract
Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance, which is a key virulence trait of this uropathogen. Each protomer of the enzyme has an N-terminal trimerization stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, two PmScsC variants, PmScsCΔN and PmScsCΔLinker, are characterized. PmScsCΔN is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsCΔN reported here reveals, as expected, a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant, PmScsCΔLinker, was designed to remove the 11-amino-acid linker, and it is shown that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsCΔLinker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small-angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentrations, whereas PmScsCΔLinker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase the understanding of disulfide isomerase activity, showing how (i) oligomerization, (ii) the spacing between and (iii) the dynamic motion of catalytic domains in PmScsC all contribute to its native function.
Highlights
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)
Smallangle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentrations, whereas PmScsCÁLinker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations
These findings increase the understanding of disulfide isomerase activity, showing how (i) oligomerization, (ii) the spacing between and (iii) the dynamic motion of catalytic domains in PmScsC all contribute to its native function
Summary
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). 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 unable to interact with its redox partner EcDsbD (Goldstone et al, 2001). 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). PmScsC lacking the trimerization stem (the N-terminal 41 residues; PmScsCÁN) is inactive as a disulfide isomerase but does have dithiol oxidase activity (Furlong et al, 2017). The difference in the activity profile between the native protein (disulfide isomerase) and PmScsCÁN (dithiol oxidase) was proposed to be a consequence of their different oligomeric states
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