Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins.

The photoactive Orange Carotenoid Protein (OCP) photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain is the active part of the protein, and the C-terminal domain regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins [HCPs]), paralogs of the N-terminal domain of OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized, and they proved to be very good singlet oxygen quenchers. When synthesized in Escherichia coli or Synechocystis PCC 6803, CTDHs formed dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo-CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable to do so. HCPs need the presence of CTDH to become holo-proteins. We propose that, in cyanobacteria, the CTDHs are carotenoid donors to HCPs.

The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction.

To deal with fluctuating light condition, cyanobacteria have developed a photoprotective mechanism which, under high light conditions, decreases the energy arriving at the photochemical centers. It relies on a photoswitch, the Orange Carotenoid Protein (OCP). Once photoactivated, OCP binds to the light harvesting antenna, the phycobilisome (PBS), and triggers the thermal dissipation of the excess energy absorbed. Deactivation of the photoprotective mechanism requires the intervention of a third partner, the Fluorescence Recovery Protein (FRP). FRP by interacting with the photoactivated OCP accelerates its conversion to the non-active form and its detachment from the phycobilisome. We have studied the interaction of FRP with free and phycobilisome-bound OCP. Several OCP variants were constructed and characterized. In this article we show that OCP amino acid F299 is essential and D220 important for OCP deactivation mediated by FRP. Mutations of these amino acids did not affect FRP activity as helper to detach OCP from phycobilisomes. In addition, while mutated R60L FRP is inactive on OCP deactivation, its activity on the detachment of the OCP from the phycobilisomes is not affected. Thus, our results demonstrate that FRP has two distinct activities: it accelerates OCP detachment from phycobilisomes and then it helps deactivation of the OCP. They also suggest that different OCP and FRP amino acids could be involved in these two activities.

Different Functions of the Paralogs to the N-Terminal Domain of the Orange Carotenoid Protein in the Cyanobacterium Anabaena sp. PCC 7120.

The photoactive Orange Carotenoid Protein (OCP) is involved in cyanobacterial photoprotection. Its N-terminal domain (NTD) is responsible for interaction with the antenna and induction of excitation energy quenching, while the C-terminal domain is the regulatory domain that senses light and induces photoactivation. In most nitrogen-fixing cyanobacterial strains, there are one to four paralogous genes coding for homologs to the NTD of the OCP. The functions of these proteins are unknown. Here, we study the expression, localization, and function of these genes in Anabaena sp. PCC 7120. We show that the four genes present in the genome are expressed in both vegetative cells and heterocysts but do not seem to have an essential role in heterocyst formation. This study establishes that all four Anabaena NTD-like proteins can bind a carotenoid and the different paralogs have distinct functions. Surprisingly, only one paralog (All4941) was able to interact with the antenna and to induce permanent thermal energy dissipation. Two of the other Anabaena paralogs (All3221 and Alr4783) were shown to be very good singlet oxygen quenchers. The fourth paralog (All1123) does not seem to be involved in photoprotection. Structural homology modeling allowed us to propose specific features responsible for the different functions of these soluble carotenoid-binding proteins.

Regulation of Orange Carotenoid Protein Activity in Cyanobacterial Photoprotection.

Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP(r)) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP(r). This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP(r), enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm.

PHOTOSYNTHESIS. A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection.

Pigment-protein and pigment-pigment interactions are of fundamental importance to the light-harvesting and photoprotective functions essential to oxygenic photosynthesis. The orange carotenoid protein (OCP) functions as both a sensor of light and effector of photoprotective energy dissipation in cyanobacteria. We report the atomic-resolution structure of an active form of the OCP consisting of the N-terminal domain and a single noncovalently bound carotenoid pigment. The crystal structure, combined with additional solution-state structural data, reveals that OCP photoactivation is accompanied by a 12 angstrom translocation of the pigment within the protein and a reconfiguration of carotenoid-protein interactions. Our results identify the origin of the photochromic changes in the OCP triggered by light and reveal the structural determinants required for interaction with the light-harvesting antenna during photoprotection.

Biosynthesis of soluble carotenoid holoproteins in Escherichia coli.

Carotenoids are widely distributed natural pigments that are excellent antioxidants acting in photoprotection. They are typically solubilized in membranes or attached to proteins. In cyanobacteria, the photoactive soluble Orange Carotenoid Protein (OCP) is involved in photoprotective mechanisms as a highly active singlet oxygen and excitation energy quencher. Here we describe a method for producing large amounts of holo-OCP in E.coli. The six different genes involved in the synthesis of holo-OCP were introduced into E. coli using three different plasmids. The choice of promoters and the order of gene induction were important: the induction of genes involved in carotenoid synthesis must precede the induction of the ocp gene in order to obtain holo-OCPs. Active holo-OCPs with primary structures derived from several cyanobacterial strains and containing different carotenoids were isolated. This approach for rapid heterologous synthesis of large quantities of carotenoproteins is a fundamental advance in the production of antioxidants of great interest to the pharmaceutical and cosmetic industries.

Msx1 and Msx2 act as essential activators of Atoh1 expression in the murine spinal cord.

Dorsal spinal neurogenesis is orchestrated by the combined action of signals secreted from the roof plate organizer and a downstream transcriptional cascade. Within this cascade, Msx1 and Msx2, two homeodomain transcription factors (TFs), are induced earlier than bHLH neuralizing TFs. Whereas bHLH TFs have been shown to specify neuronal cell fate, the function of Msx genes remains poorly defined. We describe dramatic alterations of neuronal patterning in Msx1/Msx2 double-mutant mouse embryos. The most dorsal spinal progenitor pool fails to express the bHLH neuralizing TF Atoh1, which results in a lack of Lhx2-positive and Barhl2-positive dI1 interneurons. Neurog1 and Ascl1 expression territories are dorsalized, leading to ectopic dorsal differentiation of dI2 and dI3 interneurons. In proportion, the amount of Neurog1-expressing progenitors appears unaffected, whereas the number of Ascl1-positive cells is increased. These defects occur while BMP signaling is still active in the Msx1/Msx2 mutant embryos. Cell lineage analysis and co-immunolabeling demonstrate that Atoh1-positive cells derive from progenitors expressing both Msx1 and Msx2. In vitro, Msx1 and Msx2 proteins activate Atoh1 transcription by specifically interacting with several homeodomain binding sites in the Atoh1 3' enhancer. In vivo, Msx1 and Msx2 are required for Atoh1 3' enhancer activity and ChIP experiments confirm Msx1 binding to this regulatory sequence. These data support a novel function of Msx1 and Msx2 as transcriptional activators. Our study provides new insights into the transcriptional control of spinal cord patterning by BMP signaling, with Msx1 and Msx2 acting upstream of Atoh1.