Crystal structure of plasmoredoxin, a redox-active protein unique for malaria parasites

Abstract

Plasmoredoxin is a 22 ​kDa thiol-disulfide oxidoreductase involved in cellular redox regulatory processes and antioxidant defense. The 1.6 ​Å structure of the protein, solved via X-ray crystallography, adopts a modified thioredoxin fold. The structure reveals that plasmoredoxin, unique for malarial parasites, forms a new subgroup of thioredoxin-like proteins together with tryparedoxin, unique for kinetoplastids. Unlike most members of this superfamily, Plrx does not have a proline residue within the CxxC redox motif. In addition, the Plrx structure has a distinct C-terminal domain. Similar to human thioredoxin, plasmoredoxin forms monomers and dimers, which are also structurally similar to the human thioredoxin dimer, and, as in humans, plasmoredoxin is inactive as a dimer. Monomer-dimer equilibrium depends on the surrounding redox conditions, which could support the parasite in reacting to oxidative challenges. Based on structural considerations, the residues of the dimer interface are likely to interact with target proteins. In contrast to human and Plasmodium falciparum thioredoxin, however, there is a cluster of positively charged residues at the dimer interface of plasmoredoxin. These intersubunit (lysine) residues might allow binding of the protein to cellular membranes or to plasminogen. Malaria parasites lack catalase and glutathione peroxidase and therefore depend on their other glutathione and thioredoxin-dependent redox relays. Plasmoredoxin could be part of a so far unknown electron transfer system that only occurs in these parasites. Since the surface charge of plasmoredoxin differs significantly from other members of the thioredoxin superfamily, its three-dimensional structure can provide a model for designing selective redox-modulatory inhibitors.

Keywords: Antioxidants; Bacteroides fragilis, Bf; Crithidia fasciculata, Cf; Disulfide bonds; Human, h; Leishmania major, Lm; Monomer–dimer population; Plasmodium falciparum; Plasmodium falciparum, Pf; Plasmoredoxin; Redox; Thioredoxin; Trx, thioredoxin; Trypanosoma brucei, Tb; Wuchereria bancrofti, Wb; amino acids, aa; glutaredoxin, Grx; glutathione (reduced / oxidized), GSH / GSSG; glutathione reductase, GR; peroxiredoxin Prx, either 2-Cys-Prx or Prx1m; plasmoredoxin, Plrx; thioredoxin reductase, TrxR; trypanothione, Try (reduced, oxidized); tryparedoxin reductase, TR; tryparedoxin, Txn.

Graphical abstract

Fig. 1

Overall structure of the Plrx monomer. The canonical thioredoxin fold is colored orange; the hydrogen bonds between the strands are indicated by blue lines.the active site residues (W59, C60, K61, Y62, C63) and the positively charged residues R89, K61, K116, K117 and K121 are shown as sticks, The insertion, which is not present in classic thioredoxin structures, is stained green, the N-terminal and the C-terminal domaine are colored gold and dark red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Superpositions of (A) PfPlrx monomer structures. The structure that resulted from the hexagonal crystal is colored light blue; the four structures of the monoclinic crystal are colored dark blue, orange, green, and red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Structure variations in the thioredoxin superfamily. The C-terminal domain is colored dark red, and if present, the N-terminal domain and insertion are highlighted in gold and green, respectively. The cysteine in the active site, which forms a mixed disulfide with the target enzyme, is labeled. (a) Monomer structure of PfTrx (2mmo). (b) Dimer structure of PfPrx1m (2c0d), one subunit is stained purple, the other light purple. (c) Monomer structure of PfPlrx. (d) Monomer structure of LmTxn (3s9f) (e) Monomer structure of wbTrx (4fyu). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Putative binding sites in PfPlrx or LmTxn. (a) Surface representation of PfPlrx; residues E93 and R89 interact via a salt bridge. (b) Surface representation of LmTxn (3s9f). The molecular surfaces are color-coded according to electrostatic potential (red −10 to blue 10). (Chimera package). For orientation, some residues including the active site WCxxC are labeled. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Elution profile of PfPlrx depending on its oxidization state. In size-exclusion chromatography (SEC), PfPlrx exhibited considerably different elution profiles depending on its oxidization state. PfPlrx, which was reduced by adding Tris(2-carboxyethyl)phosphine (TCEP) (black dashed line), displayed a single peak at 18.5 ​kDa, which is in close agreement with the calculated weight of the monomeric protein of 22 ​kDa. PfPlrx that was at least partially oxidized due to a lack of TCEP eluted in two main peaks. One peak corresponded to the weight of the monomer, and the second was equivalent to the dimeric protein. The scales vary due to different amounts of protein used in both experiments.

Fig. 6

Monomer contacts in the asymmetric unit of the monoclinic crystals. Dimer AC (yellow, orange) and its relevant interface residues are drawn with sticks and colored according to the subunit color. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Interfaces (A) Superimposition of the PfPlrx-with the hTrx—dimer. The Plrx dimer is shown in the same orientation as in Fig. 6. The two subunits of PfPlrx or hTrx (1eru) are colored gold and red, or purple and pink, respectively. Active site cysteine residues and cysteine residues involved in the dimer interface are labeled. Residues of the second subunit are marked with an apostrophe. (B) Superimpostion of the PfPlrx-dimer with the PfTrx-PfTrxR -complex (4J57). For clarity only one subunit (gold) of the Plrx dimer is shown, PfTrx is colored green and PfTrxR grey, active sites are marked by C60 (Plrx) and C30/C540 (PfTrx-PfTrxr). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Comparison of the charge distribution in the dimer interface of PfPlrx and hTrx. To show the interaction surface of the two subunits, we rotated the dimer by 85°, putting the surface of subunit C in front of the surface of subunit A. We therefore show only subunit A. This dimer position is then turned by 180°, consequently subunit C is now on the bottom, and we display subunit C. Panel A shows the surfaces of the PfPlrx dimer AC. Panel B shows the surfaces of the hTrx dimer.