Crystal structures of barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 reveal features involved in protein recognition and possibly in discriminating the isoform specificity

Abstract

H-type thioredoxins (Trxs) constitute a particularly large Trx sub-group in higher plants. Here, the crystal structures are determined for the two barley Trx h isoforms, HvTrxh1 and HvTrxh2, in the partially radiation-reduced state to resolutions of 1.7 A, and for HvTrxh2 in the oxidized state to 2.0 A. The two Trxs have a sequence identity of 51% and highly similar fold and active-site architecture. Interestingly, the four independent molecules in the crystals of HvTrxh1 form two relatively large and essentially identical protein-protein interfaces. In each interface, a loop segment of one HvTrxh1 molecule is positioned along a shallow hydrophobic groove at the primary nucleophile Cys40 of another HvTrxh1 molecule. The association mode can serve as a model for the target protein recognition by Trx, as it brings the Met82 Cgamma atom (gamma position as a disulfide sulfur) of the bound loop segment in the proximity of the Cys40 thiol. The interaction involves three characteristic backbone-backbone hydrogen bonds in an antiparallel beta-sheet-like arrangement, similar to the arrangement observed in the structure of an engineered, covalently bound complex between Trx and a substrate protein, as reported by Maeda et al. in an earlier paper. The occurrence of an intermolecular salt bridge between Glu80 of the bound loop segment and Arg101 near the hydrophobic groove suggests that charge complementarity plays a role in the specificity of Trx. In HvTrxh2, isoleucine corresponds to this arginine, which emphasizes the potential for specificity differences between the coexisting barley Trx isoforms.

Figure 1.

Amino acid sequences and crystal structures of HvTrxh1 and HvTrxh2. (A) Multiple sequence alignment of HvTrxh1, HvTrxh2, and selected h-type Trxs. HvTrxh1 (Q7XZK3), HvTrxh2 (Q7XZK2), AtTrxh1 (A. thaliana Trx h-type 1, P29448), AtTrxh2 (A. thaliana Trx H-type 2, Q38879), AtTrxh3 (A. thaliana Trx h-type 3, Q42403), AtTrxh4 (A. thaliana Trx h-type 4, Q39239) AtTrxh5 (A. thaliana Trx H-type 5, Q39241), CrTrxh (C. reinhardtii Trx-h, P80028), and PtTrxh (P. tremula Trx-h, Q8S3L3) were aligned using ClustalW at the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw/). The redox-active–cysteine pair is indicated with bold red letters. Positions of the HvTrxh1 loop segments, Trp39-Pro42, Ala81-Pro83, and Val98-Gly100, are indicated with green boxes. The position of the catalytic aspartate (Asp34 in HvTrxh1) is indicated with a black box. Amino acids identical to those in HvTrxh1 are indicated with a gray background. The indication of secondary-structure elements and residue numbering are based on the structure of HvTrxh1 molecule A. (B,C) Cartoon display of the crystal structures of HvTrxh1 (B) and HvTrxh2 (C) colored magenta and green, respectively. (D) Structural alignment of HvTrxh1 (magenta) and HvTrxh2 (green) shown as Cα traces.

Figure 2.

Active-site architecture of HvTrxh1 (A) and HvTrxh2 (B). Carbon, nitrogen, oxygen, and sulfur atoms are colored green, blue, red, and yellow, respectively. The 2F _o–F _c electron density maps are presented as a gray isosurface mesh at the 1.0σ level. The water molecule buried in the internal cavity is shown as a sphere and is modeled in two alternative positions in HvTrxh1. For the HvTrxh2 structure, Cys46 and Cys49 are shown only in the reduced conformations, although they are also modeled in the oxidized conformations.

Figure 3.

Radiation disruption of the active-site disulfide in HvTrxh2. Stereo images of HvTrxh2_OX (A) and HvTrxh2_RED2, 80% reduced (B), showing a close-up view of segment Cys46−Cys49. The 2F _o–F _c electron density maps are shown as gray isosurface mesh at the 1.0σ level. (C) HvTrxh2_RED1 with the 1/V∑ | F(h)_RED1 − F(h)_RED2 | e^{i ϕRED2(h)} e ^{−2πi(h − r)} difference electron density contoured at the 7σ level. Significant negative (red) density represents areas where the HvTrxh2_RED2 structure has a higher level of electron density than the HvTrxh2_RED1 structure, and positive (green) electron density, the opposite. For HvTrxh2_RED1 and HvTrxh2_RED2 structures, Cys46 and Cys49 are modeled and shown in two alternative conformations (for oxidized and reduced states).

Figure 4.

Substrate-binding loop motif in HvTrxh1 (A) and HvTrxh2 (B). Cartoon display of HvTrxh1 with sticks showing the loop segments Trp39-Pro42, Ala81-Pro83, and Val98-Gly100 (A, left). The vacuum electrostatic potential surface of HvTrxh1 (from the same angle as the left image) with the positions for Glu80 and Arg101 indicated (A, right). HvTrxh2 is presented accordingly (B). Met82 of HvTrxh1 and Met88 of HvTrxh2 are modeled and shown in two alternative conformations. For the HvTrxh2 structure, Cys46 is only shown in the reduced conformation, although it was also modeled in the oxidized conformation.

Figure 5.

Features of the HvTrxh1 crystal dimer. (A) Cartoon display shows the dimerization of HvTrxh1 molecules A (green) and D (yellow). Residues Trp39−Pro42, Ala81−Pro83, and Val98−G100 that constitute the substrate-binding loop motif are colored red. (B) Stereoview of the interface between HvTrxh1 molecules A and D. Cartoon display is shown transparently and colored green (molecule A) and yellow (molecule D). Key residues are shown in stick representation and labeled in black (molecule A) and red (molecule D), respectively. Hydrogen bonds are shown as dashed yellow lines. Glu80 (molecule D) is modeled in two alternative conformations. (C) The image is identical to the left image of Figure 5B, except that the vacuum electrostatic potential surface is shown for HvTrxh1 molecule A. (D) Cartoon display shows the interface between HvTrxh2 (blue) and BASI (pink) in the structure of HvTrxh2–BASI complex (Maeda et al. 2006). Key residues are shown in stick representations and labeled with black (HvTrxh2) and red (BASI) letters. The vacuum electrostatic potential surface is shown for HvTrxh2 in the right image.