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Structure of the Thioredoxin-Like Domain of Yeast Glutaredoxin 3

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Structure of the Thioredoxin-Like Domain of Yeast Glutaredoxin 3 University of South Carolina Scholar Commons Faculty Publications Chemistry and Biochemistry, Department of 8-13-2008 Structure of the Thioredoxin-like Domain of Yeast Glutaredoxin 3 Lydia M. Gibson University of South Carolina - Columbia Nin N. Dingra University of South Carolina - Columbia Caryn E. Outten University of South Carolina - Columbia, [email protected] Lukasz Lebioda University of South Carolina - Columbia, [email protected] Follow this and additional works at: https://scholarcommons.sc.edu/chem_facpub Part of the Chemistry Commons Publication Info Published in Acta Crystallographica Section D: Biological Crystallography, Volume 64, Issue 9, 2008, pages 927-932. © Acta Crystallographica Section D: Biological Crystallography 2008, International Union of Crystallography. This Article is brought to you by the Chemistry and Biochemistry, Department of at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. research papers Acta Crystallographica Section D Biological Structure of the thioredoxin-like domain of yeast Crystallography glutaredoxin 3 ISSN 0907-4449 Lydia M. Gibson,a Nin N. Yeast glutaredoxin 3 (Grx3) is a cytosolic protein that Received 20 May 2008 Dingra,a Caryn E. Outtena,b* and regulates the activity of the iron-responsive transcriptional Accepted 11 July 2008 Lukasz Lebiodaa,b* activator Aft1. This member of the monothiol glutaredoxin family contains a thioredoxin-like domain and a glutaredoxin- PDB Reference: thioredoxin- like domain, which both possess a monothiol active site. The like domain of yeast gluta- a Department of Chemistry and Biochemistry, crystal structure of the thioredoxin-like domain has been redoxin 3, 3d6i, r3d6isf. University of South Carolina, Columbia, ˚ SC 29208, USA, and bCenter for Colon Cancer determined at 1.5 A resolution and represents the first Research, University of South Carolina, published structure of this domain for the monothiol Columbia, SC 29208, USA glutaredoxin family. The loop containing the signature motif WAxxC is partially disordered, indicating a greater degree of flexibility in this region compared with classical dithiol Correspondence e-mail: [email protected], thioredoxins with a WCGPC active-site motif. [email protected] 1. Introduction Thioredoxins (Trxs) and glutaredoxins (Grxs) are small oxidoreductases (usually 9–15 kDa) that are found in all types of organisms. Both proteins are members of the thioredoxin- fold superfamily and typically catalyze thiol–disulfide exchange reactions. The classical dithiol Trxs utilize a highly conserved WCGPC active-site motif to reduce disulfides in substrate proteins. The dithiol Grxs possess a CPYC active- site motif and primarily catalyze glutathionylation and deglutathionylation of target proteins (Berndt et al., 2008). A subfamily of Grxs were recently identified that are char- acterized by a conserved monothiol CGFS active-site motif (Herrero & de la Torre-Ruiz, 2007). Saccharomyces cerevisiae has three Grx-like proteins that belong to this subclass: yGrx3, yGrx4 and yGrx5. yGrx5 contains a single Grx-like domain and is localized to the mitochondrial matrix, where it plays an ill-defined role in Fe–S cluster biogenesis (Rodriguez- Manzaneque et al., 2002). However, recent evidence from studies of yGrx5 homologues in chloroplasts suggest that this protein may act as a scaffold for Fe–S cluster assembly and transfer (Bandyopadhyay et al., 2008). Homologues of yGrx5 with a single Grx-like domain are found in both prokaryotes and eukaryotes (Herrero & de la Torre-Ruiz, 2007). Yeast Grx3 and Grx4 are highly homologous proteins that possess both an N-terminal Trx-like domain and a C-terminal Grx-like domain (Herrero & de la Torre-Ruiz, 2007). Both proteins are cytosolic and are required for iron-responsive inhibition of the transcriptional activator Aft1 (Kumanovics et al., 2008; Ojeda et al., 2006; Pujol-Carrion et al., 2006). The conserved cysteine in the CGFS active site located in the Grx- # 2008 International Union of Crystallography like domain of yGrx3 and yGrx4 is required for interaction Printed in Singapore – all rights reserved with Aft1 and regulation of its activity (Ojeda et al., 2006). The Acta Cryst. (2008). D64, 927–932 doi:10.1107/S0907444908021641 927 research papers Table 1 BL21 (DE3) (Novagen) as described previously (Kumanovics Crystallographic data and refinement statistics. et al., 2008). Briefly, transformants were grown at 310 K until Values in parentheses are for the outer shell. OD600 reached 0.6 and were induced with 1 mM isopropyl -d- X-ray source APS 22ID (SER-CAT) 1-thiogalactopyranoside. Cells were harvested 3 h after Wavelength (A˚ ) 1.0000 induction and stored at 193 K. Cells were lysed using three No. of frames (high pass/low pass) 400/360 freeze–thaw cycles and soluble protein was extracted with Oscillation range (high pass/low pass) () 0.5/0.5 Crystal-to-detector distance 200/300 50 mM Tris–HCl pH 8.0, 5 mM DTT. The extract was loaded (high pass/low pass) (mm) onto a DEAE anion-exchange column (GE Healthcare) Temperature (K) 100 equilibrated with 25 mM Bis-Tris pH 6.0, 5 mM DTT and Space group P21 Unit-cell parameters eluted with a salt gradient. The fractions containing yGrx3 a (A˚ ) 47.91 were further purified by gel filtration using a Superdex 75 b (A˚ ) 48.61 column (GE Healthcare) in 50 mM Tris–HCl pH 8.0, 150 mM c (A˚ ) 55.29 () 116.8 NaCl, 5 mM DTT. The final pure protein was concentrated Volume (A˚ 3) 114980 with the addition of 5% glycerol and stored at 193 K. The Solvent content (%) 46 typical yield was 30 mg of protein per litre of cell culture. Matthews coefficient (A˚ 3 DaÀ1)2.3 Mosaicity () 0.44 Protein purity was checked by SDS–PAGE and MALDI–TOF, Resolution range (A˚ ) 32.1–1.50 (1.55–1.50) indicating a single species corresponding to 28 120 kDa (the Average redundancy 3.5 (1.9) calculated molecular weight is 28 130 Da upon removal of Average I/(I) 18.7 (2.6) Total no. of reflections 121469 (1744) Met36). No. of unique reflections 35147 (1646) Completeness (%) 95.9 (69.1) Total linear Rmerge (%) 6.2 (34.7) R value (REFMAC5) (%) 22.5 (30.7) Rfree value (REFMAC5) (%) 24.9 (30.4) No. of water molecules 145 Average B factors (chain A/chain B)(A˚ 2) 17.6/17.4 Ramachandran statistics Residues in most favored regions (%) 92.7 Residues in additional allowed regions (%) 5.8 Residues in generously allowed regions (%) 1.6 Residues in disallowed regions (%) 0 Trx-like domains have a WAxPC motif in place of the signa- ture WCGPC active-site motif found in classical dithiol Trxs. Monothiol Grxs with this hybrid Trx-Grx domain architecture are only found in eukaryotes (Herrero & de la Torre-Ruiz, 2007). In general, the Grx-like domains of this protein family are more highly conserved than the Trx-like domains (Isakov et al., 2000). The human homologue of yGrx3/4 (termed PICOT for PKC-interacting cousin of Trx) has an N-terminal Trx-like domain followed by two Grx-like domains (Isakov et al., 2000). The Trx-like domain specifically interacts with protein kinase C- and is proposed to play a role in regulating the function of this kinase and its downstream targets (Witte et al., 2000). Here, we present the crystal structure of the N-terminal Trx- like domain of yGrx3 determined at 1.5 A˚ resolution. This report represents the first published structural information for the Trx-like domain for any member of the monothiol glutaredoxin/PICOT family. Figure 1 2. Materials and methods Comparison of yGrx3 Trx domain and hTrx. (a) Sequence alignment of the crystallized form of yGrx3 (amino acids 37–148) and the complete 2.1. Protein expression and crystallization sequence of hTrx. The hTrx active-site motif and cis-Pro75 are indicated The yGrx3 open reading frame containing both the Trx-like by asterisks. (b) Superposition of yGrx3 Trx domain (green) with Cys72 in green atom type and hTrx (PDB code 1ert; blue) with Cys32 and Cys35 in and Grx-like domains but lacking the first 35 amino acids (Á1– blue atom type. The positions of the amino-acid inserts are marked by 35) was cloned into pET21a and expressed in Escherichia coli arrows. 928 Gibson et al. Thioredoxin-like domain of yeast glutaredoxin 3 Acta Cryst. (2008). D64, 927–932 research papers The final crystallization conditions were optimized from a 1.0000 A˚ wavelength in two passes: a first pass with a crystal- initial crystallization-condition leads obtained using Crystal to-detector distance of 200 mm and a second pass with a Screen Lite from Hampton Research. Crystals of yGrx3 were crystal-to-detector distance of 300 mm, both using a 0.5 grown under high-salt conditions by the hanging-drop method oscillation range. The data were indexed and processed using at 300 K. Briefly, 2 ml protein solution (11 mg mlÀ1 protein in the HKL-2000 software (Otwinowski & Minor, 1997); 50 mM Tris pH 8.0 and 5 mM DTT) was mixed with 2 ml processing parameters and statistics are summarized in Table 1. reservoir solution (0.1 M MOPS pH 7.5, 20 mM ME and 68% saturated ammonium sulfate). 2.3. Structure determination and refinement The structure was solved by molecular replacement using 2.2. Data collection and processing AMoRe (Navaza, 1994) from the CCP4 system (Collaborative A yGrx3 crystal was transferred to cryosolvent (60% Computational Project, Number 4, 1994) with the structure of ammonium sulfate, 20 mM ME, 0.1 M MOPS and 20% the reduced form of human thioredoxin (hTrx) as the search ethylene glycol) for a few seconds and subsequently flash- model (PDB code 1ert; Weichsel et al., 1996).
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