Mutagenesis and recombinant expression of active site variants of the Arabidopsis CC-type glutaredoxin GRX480 reveal an altered protein expression profile in E.coli and changes in structural integrity of the GRX480 protein and its associated complex formation.

Abstract

Stress conditions such as high temperature, drought, high salinity, metal stress, and pathogenic infection significantly increase production of reactive oxygen species (ROS) in the cell. Glutaredoxins (GRXs) are small redox proteins that possess conserved cysteine (C or Cys) residues in their active site and exhibit oxidoreductase activity to protect vital proteins from oxidative damage. CPYC and CGFS type GRXs can be found in humans, yeast, E.coli, and both lower and higher plants. The CC-type class is entirely plant-specific and is thought to have emerged with the evolution of intricate signalling mechanisms involved in plant disease resistance and floral complexity. The Arabidopsis CC-type glutaredoxin, GRX480, possesses a Cys-Cys-Met-Cys (CCMC) active site and is thought to participate in the salicylic acid (SA) mediated pathways involved in plant Systemic Acquired Resistance (SAR) to confer immediate and long term resistance to biotrophic pathogens. The structural and stoichiometric properties of the GRX480 protein remain uncharacterized and the mystery surrounding the role of the conserved cysteine residue (CCMC) in CC-type glutaredoxins make this protein a prime candidate for mutagenesis studies. The AtGRX480 genetic sequence was codon optimized to allow for improved recombinant expression in E.coli cell cultures. Single (SCMC, CSMC, CCMS), double (SSMC, CSMS, SCMS), and triple (SSMS) GRX480 active site variants were created by site directed mutagenesis and a Strep II tag was added to the C-terminal end of the protein for isolation purposes. Recombinant expression of these proteins in E.coli DE3 cells caused a drastic decrease in total protein concentration when compared to untransformed cultures. Wildtype GRX480 exhibited a 6.5 fold decrease while the active site variants exhibited fold reductions within a range of 1.9 to 12.7, the CSMS and CSMC variants being the lowest and highest fold reductions respectively. All recombinant protein expression caused a decrease in protein bands within the 36-32, 20, 16, and 12 kDa range of the native protein expression profile of E.coli cultures. When isolating the GRX480 proteins on the FPLC adapted Strep-column, a single injection of crude protein solution was ineffective in isolating sufficient amounts of protein to be examined by SDS-PAGE and immunoblot analysis. The utilization of a multiple injection method drastically improved GRX480 protein isolate yield from the column, however the majority of the protein remained bound to the column as desthiobiotin was demonstrated to be a poor eluting substrate. Examination of this isolate by gel filtration chromatography, SDS PAGE, and immunoblot revealed that wildtype GRX480 (CCMC) and the SCMC, CSMS, SSMC, and SSMS variants form a tetrameric complex in vitro. The SCMS, CSMS, and CCMS variants exhibited formation of an extremely large complex, a trimeric complex, or were not observable as an intact protein during gel filtration respectively. The SCMC, CCMS, CSMC, and CSMS variants displayed a distinct degradation pattern in the N-terminal region of the protein with the CSMC and CSMS variants possessing an additional distinct degradation band. Submission of the GRX480 amino acid sequence to the I-TASSER (Iterative Threading ASSEmbly Refinement) server revealed alternative GSH binding amino residues other than the N-terminal active site cysteine. High sequence homology and predicted structural similarities of GRX480 to monothiol and dithiol glutaredoxins known to be involved in iron-sulfur [FeS]-cluster biosynthesis or [Fe-S] mediated redox sensing were also identified by I-TASSER. These results together offer novel and previously unreported features of the GRX480 protein in terms of complex formation, the roles of the active site cysteine residues, and the observed changes in native E.coli protein concentrations with recombinant expression of the GRX480 protein.