Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP

Abstract

Fasciclins (FAS1) are ancient adhesion protein domains with no common small ligand binding reported. A unique microalgal FAS1-containing astaxanthin (AXT)-binding protein (AstaP) binds a broad repertoire of carotenoids by a largely unknown mechanism. Here, we explain the ligand promiscuity of AstaP-orange1 (AstaPo1) by determining its NMR structure in complex with AXT and validating this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis. α1-α2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, forming a hydrophobic tunnel, too short to cap the AXT β-ionone rings and dictate specificity. AXT-contacting AstaPo1 residues exhibit different conservation in AstaPs with the tentative carotenoid-binding function and in FAS1 proteins generally, which supports the idea of AstaP neofunctionalization within green algae. Intriguingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids under identical conditions. These structure-activity relationships provide the first step towards the sequence-based prediction of the carotenoid-binding FAS1 members.

Fig. 1. Localization of the carotenoid-binding region of AstaPo1.

a Structural formulae of several carotenoids which can be bound by AstaPo1. AXT astaxanthin, CAN canthaxanthin, ZEA zeaxanthin, βCar β-carotene. b The domain structure of AstaPo1 showing the location of the predicted FAS1-like domain and the flanking N- and C-terminal regions. Note that the AstaPo1 preprotein contains an N-terminal signal peptide (residues 1–20), which was omitted in our construct as this peptide hampers the expression and purification of the soluble protein. Instead, the N terminus of the protein carried artificial residues ¹⁷GPHM²⁰ left after the His₆-tag removal. c, d Analytical SEC profiles with diode-array detection, revealing the effect of AstaPo1 truncations on the size of the protein monomer (c) and its absorbance spectrum (d). The apparent M_w values determined from column calibration are indicated in kDa. e Intrinsic Trp fluorescence spectra of AstaPo1 showing the effect of ZEA binding to the wild-type and truncated AstaPo1. The excitation wavelength was 297 nm. f DSC thermograms showing the effect of carotenoid binding and truncation of the N- and C-terminal regions on the thermal stability of AstaPo1. T_m values are indicated as determined from the maxima of the peaks. The heating rate was 1 °C per min. The insert shows the color of the AstaPo1 samples with ZEA or CAN used.

Fig. 2. Spatial structure of the AstaPo1(AXT) complex.

a Chemical structure of the AXT stereoisomer used for NMR structure determination, with the indication of atom numbering. Asterisks indicate the chiral centers. Oxygenated groups are in red. b Twenty NMR structures of AstaPo1(AXT) complex with the lowest restraint violations, shown superimposed over the backbone atoms of secondary structure elements (except for the C-terminal helix α6, residues 191–204) in two orthogonal views. The unstructured tails are hidden for clarity. Elements of the secondary structure are labeled. The AXT molecule is shown by orange sticks. c A single representative full-atom NMR structure of AstaPo1(AXT) provides an excellent fit to the SAXS data collected for the AstaPo1(ZEA) complex (R_g of the model is 2.09 nm, χ² = 1.5; at 7 mg mL⁻¹). R_g of the twenty NMR full-atom structural models ranged from 1.90–2.56 nm. Secondary structures are color-coded (red—α-helices, blue—β-strands, and gray—unstructured regions). d The fits to the SAXS data from the best-fitting NMR structure of the full-atom AstaPo1(AXT) complex, from the AstaPo1 FAS1 domain (residues 39–204; model R_g 1.85 nm), from the Bombyx mori Carotenoid-Binding Protein (model R_g 1.85 nm), and from the Synechocystis Orange Carotenoid Protein (model R_g 2.05 nm). The quality of the fits is indicated as χ² for each case. The insert shows the dimensionless Kratky plot for the AstaPo1(ZEA) complex. Dashed lines show the position of the maximum for the rigid sphere, which is given for reference.

Fig. 3. AXT binding mode and AstaPo1–pigment interactions.

a–e Different views of the carotenoid binding tunnel of AstaPo1. The coloring scheme in panel a is indicated. b, e Polar headgroups of AXT exiting the protein molecule from both sides of the carotenoid-binding tunnel. The Connolly surface with solvent radius 1.4 Å of AstaPo1 and the carotenoid is shown in gray and orange, respectively. c, d Polar contacts formed between the AXT polar groups and the side chain of the non-conserved Q56 residue of AstaPo1. f Amino acid conservation mapped onto the tertiary (left) and primary structure of AstaPo1 (right), according to Consurf analysis of 150 FAS1 domain-containing homologs using the scale shown in the bottom right corner (yellow color indicates positions with insufficient data). The full AstaPo1 sequence is presented for the numbering consistency (residues 1–223). Red asterisks mark the peculiar positions discussed in the text. Blue circles highlight 17 residues whose solvent-accessible surface area changes by at least 10 Ų upon AXT binding. FAS1-specific conserved motifs H1, H2, and YH are indicated by black bars. g Vis-UV CD spectra of AstaPo1(ZEA) and its truncated variant AstaPo1^41–190(ZEA) as compared with the spectra of BmCBP(ZEA) and free ZEA in methanol. The main extrema are indicated in nm. h Absorbance spectra of AstaPo1, its W79F and I176F variants purified from E. coli cells synthesizing ZEA.

Fig. 4. Insights into the carotenoid capture mechanism by AstaPo1.

a Structure of the AstaPo1(AXT) complex is colored according to the NMR data obtained for the apoform. Regions with known NMR chemical shifts, which reveal the identical structure with the AstaPo1 holoform, are colored green (Supplementary Fig. 10), and the other parts of the protein, for which no assignment could be obtained, are colored light gray. b A hypothetical carotenoid capture mechanism. Three panels represent from left to right: the CupS FAS1-containing protein (PDB ID: 2MXA) in a tentative “open-like” conformation, AstaPo1 in complex with AXT (our NMR structure), and their overlay. The structures are colored by a gradient from blue (N) to red (C), except for the conserved motifs used for structural alignment—H1 (magenta), H2 (pale cyan), and YH (pale pink). The pairwise sequence alignment of the H1 and H2 fragments is shown below (identical residues are in bold font). AXT is shown as an orange ball-and-stick model, the main secondary structure elements and the hinge loop are labeled. The proposed conformational transition from an open to a carotenoid-bound state is depicted by the arrow. c Far UV CD spectrum of the AstaPo1 apoform (black line) is shown overlaid with the average CD spectrum calculated from 20 NMR models of the holoform of the same construct (red dashed line).

Fig. 5. Carotenoid binding as neofunctionalization of AstaP orthologs.

a Multiple sequence alignment (MSA) of the FAS1 domains of the tentative carotenoid (Car) binding AstaP orthologs from Coelastrella astaxanthina (AstaPo1; BAN66287.1), Coelastrella vacuolata (CvacAstaP1, QYF06643.1; CvacAstaP2; QYF06644.1), Scenedesmus costatus (ScosAstaP, QYF06645.1), Scenedesmus sp. Oki-4N (AstaPo2-FAS1 domain 1 and AstaPo2-FAS1 domain 2, BBN91622.1; AstaP-pink1, BBN91623.1; AstaP-pink2, BBN91624.1), Scenedesmus acutus (SacutAstaP; ACB06751.1), and Synechocystis sp. PCC 6803 (SynAstaP, Uniprot P74615), shown with coloring by similarity as shades of blue. NCBI accession numbers are indicated for each sequence (except SynAstaP) in parentheses. The sequence identity of each ortholog relative to AstaPo1 is indicated in %. Seventeen positions of the AstaPo1 sequence whose solvent-accessible surface area (SASA) changed >10 Ų upon AXT binding (NMR data) are mapped on the MSA to reveal positions satisfying the criteria indicated by magenta, orange, and cyan. Note that positions of Leu57 and Trp79 of AstaPo1 are occupied by Phe residues in SynAstaP (P74615) (marked in red). b Comparison of the NMR structure of AstaPo1 complexed with AXT and the Alphafold model of Synechocystis ortholog P74615, showing the similar FAS1-like fold featuring the jaw and the tunnel. c Structure alignment of AstaPo1(AXT) complex (blue) and the Alphafold model of SynAstaP (Uniprot P74615), indicating the nearly identical tunnel lining and different tunnel exits. Note that the side chain of the non-conserved Phe58 of P74615 clashes with the carotenoid polyene, that the non-conserved Gln56 of AstaPo1 contacting the polar groups of AXT is replaced by a shorter Thr57 residue in P74615, and that a conserved Trp79 of AstaPo1 is replaced by Phe80 in P74615 (the corresponding distances are shown in a). d, e The zeaxanthin-binding capacity of AstaPo1 (50 μM) and SynAstaP (Uniprot P74615) (500 μM) analyzed by SEC of proteins purified from E. coli cells synthesizing zeaxanthin. Note that SynAstaP has an extremely low extinction coefficient at 280 nm due to the total absence of Trp and the presence of only one Tyr residue, hence it was loaded in the tenfold higher concentration. The apparent M_w of the peaks are shown. The absorbance spectra recorded during SEC runs and corresponding to the peak maxima are presented in the inserts. For SynAstaP, a similar spectrum of the protein obtained by mixing with ZEA in vitro prior to SEC is also presented. Absorbance maxima are indicated. The inserts in panel d also show the electrophoretic purity of the P74615 preparation used for the analysis and the appearance of the AstaPo1 and P74615 samples purified from ZEA-synthesizing E. coli cells. f Absorbance spectra of AstaPo1 WT and its mutant versions purified from ZEA-synthesizing E. coli cells under identical conditions. FWHM - full width at half maximum. Positions (nm) of the main spectral features are marked.