Characterization of poplar GrxS14 in different structural forms

Protein Cell 2014, 5(5):329–333 DOI 10.1007/s13238-014-0042-3 Protein & Cell LETTER Dear Editor, Glutaredoxins (Grxs) are glutathione-dependent thiol disulfide oxidoreductases of the thioredoxin family present in all organisms from bacteria to human (Noguera et al., 2005). Depending on their active site sequence, Grxs are essentially classified into three families: the dithiol Grxs, the monothiol Grxs and the CC type restricted to plants (Rouhier et al., 2008). Grxs play important biological functions in plants, such as oxidative stress responses, iron-sulfur (FeS) cluster assembly, and cell signaling, etc. (Rouhier et al., 2008). There are totally 31 Grxs isoforms in Arabidopsis thaliana, and 19 Grx isoforms in Populus trichocarpa (Rouhier et al., 2004). For Grxs of Populus trichocarpa, structures of GrxC1, GrxC4 and GrxS12 have been resolved, all belong to dithiol Grxs (Noguera et al., 2005; Feng et al., 2006; Rouhier et al., 2007; Couturier et al., 2009). The only structure available for monothiol Grxs in plants is that of Arabidopsis Grxcp, which is also called GrxS14 or CAXIP1 (Cheng and Hirschi, 2003; Li et al., 2010). It was found that Arabidopsis GrxS14 is a new class of signaling molecules in plants that can regulate the Ca2+ transport activity of CAX1 (cation exchangers) by interacting with the N-terminal region of CAX1 (Cheng and Hirschi, 2003). It was suggested that Arabidopsis GrxS14 functions to protecting cells against protein oxidative damage (Cheng et al., 2006). Both Arabidopsis and poplar GrxS14 are monothiol Grxs located in the chloroplasts, which exist as an apo form and a holo form bridged by a [2Fe-2S] cluster with two external glutathione (GSH) ligands, and they can complement a yeast grx5 mutant defective in FeS cluster assembly in vivo (Bandyopadhyay et al., 2008). It was proposed that Arabidopsis and poplar GrxS14 may function as scaffold protein for the assembly of [2Fe-2S] cluster, as GrxS14 can transfer intact cluster to physiologically relevant acceptor proteins which is regulated by GSH (Bandyopadhyay et al., 2008; Wang et al., 2012; Liu et al., 2013). Here we report the solution structure of reduced poplar GrxS14 and structure models for the non-covalent apo GrxS14 dimer and GrxS14/GSH complex, as well as the NMR characterization of holo GrxS14. The quality of the 2D 1H-15N HSQC spectrum of apo GrxS14 at 1 mmol/L concentration was very poor (Fig. S1), and very few signals could be observed in 3D triple-resonance NMR spectra. Dilution of the sample did not improve the quality of NMR spectra very significantly. Interestingly, much better NMR spectra were obtained with the addition of GSH (Fig. S1). Although apo GrxS14 appeared to be a monomer on the gel filtration column, analytical ultracentrifugation analysis showed two peaks (with molecular weight about 24 kDa and 12 kDa) for apo GrxS14 without GSH, while there was only one peak at ∼12 kDa for apo GrxS14 with GSH (Fig. S2A and S2B). All these suggest that apo GrxS14 should be in a monomer-to-dimer equilibrium, and the dimerization can be inhibited by GSH. Thus, there exists another type of GrxS14 dimer in addition to the holo GrxS14 dimer assembled with a [2Fe-2S] cluster. Based on the analytical ultracentrifugation data (Fig. S2A), the dissociation constant of GrxS14 monomer-to-dimer equilibrium is estimated to be ∼0.4 mmol/L. We determined the solution structure of reduced monomeric GrxS14 using NMR data collected on protein samples in the presence of 20 mmol/L GSH. A summary of structural restraints used in the structure calculation and statistics for the structure ensemble is listed in Table S1. Residues 5–109 of apo GrxS14 form a compact thioredoxin fold structure while the first four residues are flexible (Fig. 1A). It comprises five α-helices and four β-strands, the four β-strands constitute a mixed β-sheet as the core of structure (Fig. 1B). Helices α1 and α3 are packed on one side of the β-sheet, while α2, α4 and α5 are on the other side. The overall fold of poplar GrxS14 is similar to other Grxs. The RMSD of backbone heavy atoms in secondary structure regions is 1.7 Å between poplar and Arabidopsis GrxS14 which shares an 80% sequence identity (Figs. 1D and S3A). The relatively large RMSD between the two may be due to that the poplar apo GrxS14 was determined in the presence of GSH, while no GSH is in the crystal structure of Arabidopsis GrxS14. When comparing to dithiol Grxs, the major difference is at the loop region between β1 and α2: poplar GrxS14 contains ten amino acid residues, while only four residues in poplar GrxC1 and human Grx2 (Fig. 1C). Sequence alignment indicated that monothiol Grxs all possess a long loop in this region, whereas the dithiol Grxs usually have a short loop (Fig. S3). This long loop before the active site (CGFS) is a structural characteristic of monothiol Grxs. © The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn Protein & Cell Characterization of poplar GrxS14 in different structural forms LETTER A Lei Wang et al. B α1 α3 β4 α5 β2 β1 β3 α2 α4 Protein & Cell C D Figure 1. Solution structure and structural comparison of reduced apo GrxS14. (A) The backbone superimposition of 20 structures. (B) The ribbon representation of the mean structure. (C) Structural comparison of poplar GrxS14 (sky blue) and poplar GrxC1 (light yellow) (PDB 1Z7P, 1Z7R). Loop regions between β1 and α2 are shown as blue and yellow, respectively. (D) Structural comparison of poplar GrxS14 (light gray) and Arabidopsis GrxS14 (light blue) (PDB 3IPZ). Residues with significant combined NH chemical shift changes upon GSH binding are indicated on the structure of poplar GrxS14 by yellow color (0.05 < δ < 0.1 ppm), orange color (0.1 ≤ δ < 0.2 ppm), and orange red (δ ≥ 0.2 ppm), respectively. The side-chains of conserved active site cysteine are illustrated as ball and stick. The dimerization of apo GrxS14 was investigated using NMR spectroscopy. Comparison of 2D 1H-15N HSQC spectra of apo GrxS14 at different concentrations revealed that residues with significant concentration-dependent NH chemical shift changes are F35, Q37, K66, W71, G86, D88, I89, V91, E92 and S96, and these residues should be involved in the dimer interface (Figs. 2A and S4). Based on the structure models of apo GrxS14 dimer calculated using HADDOCK 2.0 (supplementary methods), the dimer interface is located at helices α2, α3 and α4, and loops β1-α2, α3-β3 and β4-α4 (Fig. 2B). In the dimer interface, there should be aromatic stacking interaction between the two phenyl groups of F35 since the two rings are close, and sidechains of K66 from one molecule and D88 or E92 from the other are in a distance to form salt bridges (∼2 Å) (Fig. 2B). To verify these interactions, two mutants F35A and D88A/ E92A of GrxS14 were generated. Analytical ultracentrifugation 330 analysis revealed that the amount of dimer fraction is significantly reduced for the two (...truncated)