Insights into the molecular mechanism of yellow cuticle coloration by a chitin-binding carotenoprotein in gregarious locusts.

Carotenoids are hydrophobic pigments binding to diverse carotenoproteins, many of which remain unexplored. Focusing on yellow gregarious locusts accumulating cuticular carotenoids, here we use engineered Escherichia coli cells to reconstitute a functional water-soluble ß-carotene-binding protein, BBP. HPLC and Raman spectroscopy confirmed that recombinant BBP avidly binds ß-carotene, inducing the unusual vibronic structure of its absorbance spectrum, just like native BBP extracted from the locust cuticles. Bound to recombinant BBP, ß-carotene exhibits pronounced circular dichroism and allows BBP to withstand heating (T0.5 = 68 °C), detergents and pH variations. Using bacteria producing distinct xanthophylls we demonstrate that, while ß-carotene is the preferred carotenoid, BBP can also extract from membranes ketocarotenoids and, very poorly, hydroxycarotenoids. We show that BBP-carotenoid complex reversibly binds to chitin, but not to chitosan, implying the role for chitin acetyl groups in cuticular BBP deposition. Reconstructing such locust coloration mechanism in vitro paves the way for structural studies and BBP applications.

Elements of the C-terminal tail of a C-terminal domain homolog of the Orange Carotenoid Protein determining xanthophyll uptake from liposomes.

Carotenoids perform multifaceted roles in life ranging from coloration over light harvesting to photoprotection. The Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, accommodates a ketocarotenoid vital for its function. OCP extracts its ketocarotenoid directly from membranes, or accepts it from homologs of its C-terminal domain (CTDH). The CTDH from Anabaena (AnaCTDH) was shown to be important for carotenoid transfer and delivery from/to membranes. The C-terminal tail of AnaCTDH is a critical structural element likely serving as a gatekeeper and facilitator of carotenoid uptake from membranes. We investigated the impact of amino acid substitutions within the AnaCTDH-CTT on echinenone and canthaxanthin uptake from DOPC and DMPG liposomes. The transfer rate was uniformly reduced for substitutions of Arg-137 and Arg-138 to Gln or Ala, and depended on the lipid type, indicating a weaker interaction particularly with the lipid head group. Our results further suggest that Glu-132 has a membrane-anchoring effect on the PC lipids, specifically at the choline motif as inferred from the strongly different effects of the CTT variants on the extraction from the two liposome types. The substitution of Pro-130 by Gly suggests that the CTT is perpendicular to both the membrane and the main AnaCTDH protein during carotenoid extraction. Finally, the simultaneous mutation of Leu-133, Leu-134 and Leu-136 for alanines showed that the hydrophobicity of the CTT is crucial for carotenoid uptake. Since some substitutions accelerated carotenoid transfer into AnaCTDH while others slowed it down, carotenoprotein properties can be engineered toward the requirements of applications.

Structural framework for the understanding spectroscopic and functional signatures of the cyanobacterial Orange Carotenoid Protein families.

The Orange Carotenoid Protein (OCP) is a unique photoreceptor crucial for cyanobacterial photoprotection. Best studied Synechocystis sp. PCC 6803 OCP belongs to the large OCP1 family. Downregulated by the Fluorescence Recovery Protein (FRP) in low-light, high-light-activated OCP1 binds to the phycobilisomes and performs non-photochemical quenching. Recently discovered families OCP2 and OCP3 remain structurally and functionally underexplored, and no systematic comparative studies have ever been conducted. Here we present two first crystal structures of OCP2 from morphoecophysiologically different cyanobacteria and provide their comprehensive structural, spectroscopic and functional comparison with OCP1, the recently described OCP3 and all-OCP ancestor. Structures enable correlation of spectroscopic signatures with the effective number of hydrogen and discovered here chalcogen bonds anchoring the ketocarotenoid in OCP, as well as with the rotation of the echinenone's ß-ionone ring in the CTD. Structural data also helped rationalize the observed differences in OCP/FRP and OCP/phycobilisome functional interactions. These data are expected to foster OCP research and applications in optogenetics, targeted carotenoid delivery and cyanobacterial biomass engineering.

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

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 ß-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.

Silkworm carotenoprotein as an efficient carotenoid extractor, solubilizer and transporter.

Found in many organisms, water-soluble carotenoproteins are prospective antioxidant nanocarriers for biomedical applications. Yet, the toolkit of characterized carotenoproteins is rather limited: such proteins are either too specific binders of only few different carotenoids, or their ability to transfer carotenoids to various acceptor systems is unknown. Here, by focusing on a recently characterized recombinant ~27-kDa Carotenoid-Binding Protein from Bombyx mori (BmCBP) [Slonimskiy et al., International Journal of Biological Macromolecules 214 (2022): 664-671], we analyze its carotenoid-binding repertoire and potential as a carotenoid delivery module. We show that BmCBP forms productive complexes with both hydroxyl- and ketocarotenoids - lutein, zeaxanthin, astaxanthin, canthaxanthin and a smaller antioxidant, aporhodoxanthinone, but not with ß-carotene or retinal, which defines its broad ligand specificity toward xanthophylls valuable to human health. Moreover, the His-tagged BmCBP apoform is capable of cost-efficient and scalable enrichment of xanthophylls from various crude methanolic herbal extracts. Upon carotenoid binding, BmCBP remains monomeric and shows a remarkable ability to dynamically shuttle carotenoids to biological membrane models and to unrelated carotenoproteins, which in particular makes from the cyanobacterial Orange Carotenoid Protein a blue-light controlled photoswitch. Furthermore, administration of BmCBP loaded by zeaxanthin stimulates fibroblast growth, which is attractive for cell- and tissue-based assays.

Structural basis for the carotenoid binding and transport function of a START domain.

STARD3, a steroidogenic acute regulatory lipid transfer protein, was identified as a key xanthophyll-binding protein in the human retina. STARD3 and its homologs in invertebrates are known to bind and transport carotenoids, but this lacks structural elucidation. Here, we report high-resolution crystal structures of the apo- and zeaxanthin (ZEA)-bound carotenoid-binding protein from silkworm Bombyx mori (BmCBP). Having a STARD3-like fold, BmCBP features novel elements, including the Ω1-loop that, in the apoform, is uniquely fixed on the α4-helix by an R173-D279 salt bridge. We exploit absorbance, Raman and dichroism spectroscopy, and calorimetry to describe how ZEA and BmCBP mutually affect each other in the complex. We identify key carotenoid-binding residues, confirm their roles by ZEA-binding capacity and X-ray structures of BmCBP mutants, and also demonstrate that markedly different carotenoid-binding capacities of BmCBP and human STARD3 stem from differences in the structural organization of their carotenoid-binding cavity.

Reconstitution of the functional Carotenoid-Binding Protein from silkworm in E. coli.

Natural water-soluble carotenoproteins are promising antioxidant nanocarriers for biomedical applications. The Carotenoid-Binding Protein from silkworm Bombyx mori (BmCBP) is responsible for depositing carotenoids in cocoons. This determines the silk coloration, which is relevant for sericulture for four thousand years. While BmCBP function is well-characterized by molecular genetics, its structure and carotenoid-binding mechanism remain to be studied. To facilitate this, here we report on successful production of soluble BmCBP in Escherichia coli, its purification and characterization. According to CD spectroscopy and SEC-MALS, this protein folds into a ~ 27-kDa monomer capable of dose-dependent binding of lutein, a natural BmCBP ligand, in vitro. Binding leads to a >10 nm red-shift of the carotenoid absorbance and quenches tryptophan fluorescence of BmCBP. Using zeaxanthin, a close lutein isomer that can be stably produced in engineered E.coli strains, we successfully reconstitute the BmCBP holoform and characterize its properties. While BmCBP successfully matures into the holoform, BmCBP-zeaxanthin complexes are contaminated by the apoform. We demonstrate that the yield of the holoform can be increased by adding bovine serum albumin during cell lysis and that the remaining BmCBP apoform is efficiently removed using hydroxyapatite chromatography. Bacterial production of BmCBP paves the way for its structural studies and applications.

Microalgal protein AstaP is a potent carotenoid solubilizer and delivery module with a broad carotenoid binding repertoire.

Carotenoids are lipophilic substances with many biological functions, from coloration to photoprotection. Being potent antioxidants, carotenoids have multiple biomedical applications, including the treatment of neurodegenerative disorders and retina degeneration. Nevertheless, the delivery of carotenoids is substantially limited by their poor solubility in the aqueous phase. Natural water-soluble carotenoproteins can facilitate this task, necessitating studies on their ability to uptake and deliver carotenoids. One such promising carotenoprotein, AstaP (astaxanthin-binding protein), was recently identified in eukaryotic microalgae, but its structure and functional properties remained largely uncharacterized. By using a correctly folded recombinant protein, here we show that AstaP is an efficient carotenoid solubilizer that can stably bind not only astaxanthin but also zeaxanthin, canthaxanthin, and, to a lesser extent, ß-carotene, that is, carotenoids especially valuable to human health. AstaP accepts carotenoids provided as acetone solutions or embedded in membranes, forming carotenoid-protein complexes with an apparent stoichiometry of 1:1. We successfully produced AstaP holoproteins in specific carotenoid-producing strains of Escherichia coli, proving it is amenable to cost-efficient biotechnology processes. Regardless of the carotenoid type, AstaP remains monomeric in both apo- and holoform, while its rather minimalistic mass (~ 20 kDa) makes it an especially attractive antioxidant delivery module. In vitro, AstaP transfers different carotenoids to liposomes and to unrelated proteins from cyanobacteria, which can modulate their photoactivity and/or oligomerization. These findings expand the toolkit of the characterized carotenoid binding proteins and outline the perspective of the use of AstaP as a unique monomeric antioxidant nanocarrier with an extensive carotenoid binding repertoire.