Calcium sensing via EF-hand 4 enables thioredoxin activity in the sensor-responder protein calredoxin in the green alga Chlamydomonas reinhardtii

Abstract

Calcium (Ca²⁺) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca²⁺-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca²⁺-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 Å resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca²⁺ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.

Keywords: Chlamydomonas; Förster resonance energy transfer (FRET); calcium; calredoxin (CRX); peroxiredoxin; protein crystallization; redox reaction; sensor-responder; signaling; thioredoxin.

Figure 1.

SAXS experimental data of CRX with and without Ca²⁺. (a) The experimental scattering profile of CRX at 5 mg/ml is shown in blue for the Ca²⁺-bound and in red for the Ca²⁺-free form. (b) Close-up view of CRX at 5 mg/ml, colors as in a. (c) The Guinier plot and (d) the distance distribution function P(r) (lower) of 5 mg/ml of CRX with and without Ca²⁺. (e) The dummy atom model of CRX with and without Ca²⁺ was calculated using GASBOR and DAMAVER.

Figure 2.

FRET-based analysis of Ca²⁺-induced conformational change of CRX. CFP and mVenus (YFP) were cloned N- and C-terminal of CRX and expressed heterologously in E. coli. (a) A conformational change upon Ca²⁺-binding would alter FRET from CFP to YFP as shown schematically. (b) Emission of the CRX-based Ca²⁺ reporter after excitation at 435 nm. CFP and mVenus emission peaks are indicated by blue and yellow arrows, respectively. Emission spectra with (black) and without (blue) free Ca²⁺ are shown. (c) The amount of FRET is shown as the ratio of YFP to CFP emission and in dependence on the free Ca²⁺ concentration.

Figure 3.

NMR measurements show the Ca²⁺-induced conformational change of CRX. (a) All methionines (M) in CRX are indicated as a red stick model and labeled according to their position. The ribbon represents the CRX structure (PDB ID 5E37) whose two CaM domains and TRX domain are shown in orange, pink, and blue, respectively. (b) Overlay of ¹H-¹³C HSQC spectra of [¹³C-Met]-CRX with Ca²⁺ (blue) and without Ca²⁺ (red). Numbers indicate methionine positions. (c) Average chemical shift perturbations Δδ_AV = ((Δδ¹H)² + ((0.394/1.666)Δδ¹³C)²)^1/2 of CRX Met with and without Ca²⁺. Each number represents the standard deviation of the statistical distribution of Met ¹H and ¹³C chemical shifts reported in BioMagResBank. To equalize the weights of the ¹H and ¹³C chemical shift changes for each peak, the latter was normalized by dividing it with the corresponding standard deviation ratio of the methionine chemical shift distribution found in the registered proteins. As a result, Δδ_AV values were corrected to the scale based on the ¹H chemical shift changes.

Figure 4.

CRX redox activity and interaction with PRX1 depend on EF-hand 4. Recombinant WT (black squares) and single point mutated EF-hand mutants (triangles) of CRX were reduced by E. coli thioredoxin reductase (TrxR) and NADPH in defined Ca²⁺ concentrations. 1 μm oxidized recombinant PRX1 and 80 μm H₂O₂ (a and b) or 200 μm DTNB (c) were added as a substrate for reduction by CRX. NADPH oxidation (a and b) and DTNB reduction (c) were tracked at 340 and 412 nm, respectively. To calculate the redox activity, NADPH consumption (a) or 80 s of stable slope after addition of DTNB (c) were plotted against the Ca²⁺ concentration and fitted by the Hill equation (v = (v_max × [S]^nH)/(K_Ca2+^nH + [S]^nH)). Error bars give S.D. of three to four independent measurements. Panel b shows the same data as in panel a but in semi-log representation (NADPH oxidation versus log₁₀ of Ca²⁺ concentration). Assays were modified as described in Refs. and .

Figure 5.

Chlamydomonas PRX1 crystal structure. (a) The decameric ring shape of C174S PRX1 (PDB ID 6J13). Monomers are labeled from A to E and A′ to E′. (b) model of the C. reinhardtii CRX:PRX1 complex. The C-terminal peptides of the PRX-TRX complex (PDB ID 3VWU) is shown in ball-and-stick and light-green ribbon models, respectively. PRX1 is colored in red, and mouse PRX4 is colored in light blue. CRX is colored in orange, magenta, and blue. (c) Close-up view on cross-linked region identified by MS analysis after in vitro cross-linking of recombinant CRX and PRX1. The distance between the nitrogen atoms of the cross-linked lysines Lys-273 (CRX) and Lys-94 (PRX1) is 1.9 Å. Colors are as described under b.

Figure 6.

Evolutionary origin of EF-hand containing thioredoxins. (A) Full phylogenetic tree of the protein family KOG0027, unrooted. Members from the plant kingdom (Viridiplantae) are marked green, whereas the magenta-colored clade highlights EF-hand containing thioredoxins. (B) Close-up of the EF-hand containing thioredoxins. Scale bars in A and B indicate substitutions per site. (C) Simplified representation of EF-hand containing thioredoxins. EF-hands 1, 2, 3, and 4, as well as the active core of the thioredoxin domain are highlighted in lighter gray. Sequences are presented in phylogenetic order, as in A and B. Numbers indicate the position of amino acids in CRX from Chlamydomonas as described in Ref. (dark gray sequence). The second sequence from Chlamydomonas originates from an older version of the BLAST database. Conserved amino acids involved in the proposed H-bond network are marked with a star. Note that the data are shown 5′- and 3′-truncated, zooming in on the core domains only.