Chaperone activation and client binding of a 2-cysteine peroxiredoxin

Abstract

Many 2-Cys-peroxiredoxins (2-Cys-Prxs) are dual-function proteins, either acting as peroxidases under non-stress conditions or as chaperones during stress. The mechanism by which 2-Cys-Prxs switch functions remains to be defined. Our work focuses on Leishmania infantum mitochondrial 2-Cys-Prx, whose reduced, decameric subpopulation adopts chaperone function during heat shock, an activity that facilitates the transition from insects to warm-blooded host environments. Here, we have solved the cryo-EM structure of mTXNPx in complex with a thermally unfolded client protein, and revealed that the flexible N-termini of mTXNPx form a well-resolved central belt that contacts and encapsulates the unstructured client protein in the center of the decamer ring. In vivo and in vitro cross-linking studies provide further support for these interactions, and demonstrate that mTXNPx decamers undergo temperature-dependent structural rearrangements specifically at the dimer-dimer interfaces. These structural changes appear crucial for exposing chaperone-client binding sites that are buried in the peroxidase-active protein.

Fig. 1

Cryo-EM structure of the mTXNPx_red: luciferase complex. a 2D reference-free class averages of heat-treated mTXNPx_red in the presence (left) or absence (right) of unfolded luciferase showing additional central density attributed to luciferase in top and side (arrow) views. Scale bar equals 5 nm. Cryo-EM 3D map of the mTXNPx_red:luciferase complex (b) and mTXNPx alone (c) resolved to 3.7 and 2.9 Å resolution, respectively shown at σ = 3.3. Top and side views are shown. The L. braziliensis mTXNPx crystal structure (4KB3) was docked into the EM density with additional N-terminal residues, Q31-T35, modeled for mTXNPx_red:luciferase. d The unsharpened map of the mTXNPx_red:luciferase complex displayed at threshold σ = 1.7 showing central density corresponding to bound client. e N-terminal residues Q31-T35 shown docked into the sharpened mTXNPx_red:luciferase map with approximate location of client (dashed line) adjacent Q31, M32, Y33, and R34

Fig. 2

N-terminal extensions of mTXNPx are crucial for in vitro chaperone activity. Influence of reduced ∆5mTXNPx (a) or mTXNPxY33A/mTXNPxR34A (b) on the thermal aggregation of luciferase. Native luciferase (0.1 μM) was incubated in the absence or presence of different ratios of mTXNPx at 42 °C. Light scattering (expressed in arbitrary units, A.U.) was monitored at 360 nm. All reactions were carried out in the presence of 0.2 mM DTT to maintain reduction of mTXNPx. Purified wt mTXNPx was used as positive control

Fig. 3

UV-cross-linking of mTXNPx to client proteins in vivo. a Bpa substitutions in mTXNPx. Phe and Tyr residues (indicated in red) were individually mutated to allow for the site-specific incorporation of the photo-activatable amino acid Bpa. The C_p-loop-helix, which contains the peroxidative Cys81 and which is folded when mTXNPx is reduced and decameric, is underlined in red. All regions (regions I–IV) involved in dimer–dimer interactions are underlined in blue. The mitochondrial targeting sequence is underlined in black. The targeting sequence is not present in the in vivo constructs. b In vivo cross-linking of mTXNPx-Bpa variants. E. coli expressing either wild-type mTXNPx or the mTXNPx-Bpa variants were grown at 30 °C and either kept at 30 °C or shifted to 45 °C for 30 min. Afterwards, aliquots of the cells were either left untreated (−) or were exposed (+) to UV light for 10 min to induce cross-linking. Subsequently, bacteria were lysed and cell extracts were analyzed by western blot using anti-mTXNPx antibodies. Two different exposure times are shown. The two most prominent bands corresponded to monomeric (mTXNPx) and dimeric mTXNPx-mTXNPx, respectively. Additional bands, seen as higher-migrating smear, are suggestive of mTXNPx-E. coli protein cross-links (mTXNPx-X). A representative experiment is shown (results from all experiments conducted are summarized in Supplementary Table 1). Based on the extent of higher migrating smear (i.e., cross-links) upon incubation at 30 °C and/or 45 °C, the residues were classified into three groups: Group 1: Bpa-variants that cross-link more extensively upon incubation at 45 °C compared to 30 °C (red); Group 2: Bpa-variants that show equal cross-linking at 45 °C and 30 °C (magenta); Group 3; Bpa-variants that either do not show any reproducible higher migrating smear, or show only a small number of discrete higher migrating bands (blue). Bpa-194, Bpa-221, and Bpa-222 variants either produced inconsistent results or were not reliably produced in vivo

Fig. 4

Mapping of in vivo cross-linking sites onto quaternary structure of mTXNPx. a Quaternary structure of reduced L. infantum mTXNPx_red. For simplicity, only one of the five dimers that are present in the decamer (subunit A in pink and subunit B in blue) as well as one adjacent subunit on each side (grey) are shown. Different views of the selected mTXNPx dimer are presented. Amino acids that, when replaced by Bpa, reproducibly produce higher migrating smear upon UV-cross-linking either after shift to 45 °C (Group 1, red), at both 30 °C and 45 °C (Group 2, magenta) or no shift (Group 3, blue) are shown. b Close-up view of the dimer-dimer interface and location of Group 1 residues. Group 1 residues F77 and F79 (red and grey dots) form part of the dimer-dimer interface. All other group 1 residues (in rainbow colors) are mostly buried

Fig. 5

In vitro cross-linking of mTXNPx_red-luciferase complexes. a Intra-protein (purple lines) and inter-protein (cyan lines) cross-link maps using xiNET (Supplementary Data 1). The mTXNPx sequence including the mitochondrial targeting sequence was used for the map. b Mapping of the in vitro cross-linking sites between mTXNPx_red and luciferase (shown in orange) and the in vivo cross-linking sites between mTXNPx-Bpa variants and cellular client proteins (red: Group 1 cross-links; pink: Group 2 cross-links, Fig. 4a) onto the quaternary structure of L. infantum TXNPx_red. For simplicity, only one of the five dimers that are present in the decamer (subunit A in pink and subunit B in blue) as well as one adjacent subunit on each side (grey) are shown

Fig. 6

mTXNPx decamers undergo heat-shock-induced rearrangements. a mTXNPx_red (2 µM) either alone or in complex with luciferase (0.2 µM) was incubated in the presence (+) or absence (−) of 40 mM glutaraldehyde at 30 °C or 42 °C for 10 min. The oligomeric status of mTXNPx_red was assessed by running the samples on non-reducing SDS-PAGE followed by silver staining. Dimeric mTXNPx_ox was used as control. In the absence of cross-linker, mTXNPx_red migrates in the monomeric form. b Three independent experiments were quantified using ImageJ software. Values represent means ± SD. c mTXNPx_red (10 µM) was incubated either alone or in the presence of luciferase (1 µM) for 10 min at 42 °C. One aliquot was immediately applied onto an EM grid while the other aliquot was cooled down before applying onto the grids. d Quantification of the number of decamers present in 10 micrographs for each condition +/− std is shown