Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism

Abstract

The photoswitchable orange carotenoid protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCP to the red OCP form. Cyanobacterial genomes frequently harbor, besides genes for orange carotenoid proteins (OCPs), several genes encoding homologs of OCP's N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.

Structural rearrangements in the C-terminal domain homolog of Orange Carotenoid Protein are crucial for carotenoid transfer

“…PISA suggested that only the F-type dimer has biological significance, (Δ i G P value of 0.067 for the F-type dimer interface). This finding agrees with the suggestion proposed by Moldenhauer et al (2017) 26 and Muzzopappa et al (2017) 25 that the interaction plane between the CTD(H) dimer should be at the same site as the CTD and NTD in OCP O . In contrast, a Δ i G P value of 0.518 in the case of the A-type dimer interface implies possibly crystal-stabilized interface (as a Δ i G P value  <0.5 is considered necessary for biological significance 29 ).…”

“…2a) suggested particles with a compact globular core and moderate flexibility. The Porod volume of 56 nm 3 suggests that the corresponding MW estimate (35 kDa) is close to that expected for apo-CTD dimers (37 kDa), in line with their known dimerization at concentrations above 150 µM 26,30 . Ab initio molecular shape reconstruction using DAMMIF 31 resulted in globular models with pairs of protrusions at variable positions (Fig.…”

“…The heterogeneous nature of the apo-AnaCTDH solution (Supplementary Figure 3) and this protein’s propensity to monomerize in the absence of carotenoid 25 impedes structural analysis of its non-covalently bound dimers. In contrast, individual apo-CTDs from Synechocystis OCP (carrying a Phe instead of Cys at residue position 103) efficiently dimerize without forming higher-order oligomers 26 , making apo-CTD dimers suitable for structural characterization in solution by using small-angle X-ray scattering (SAXS).…”

“…Recently, it was revealed that isolated CTD of Synechocystis OCP and CTDHs from Anabaena and Thermosynechococcus elongatus (a clade 1 CTDH) are also capable of carotenoid binding 25,26 . Both CTD and CTDH were shown to mediate carotenoid transfer to apo-OCP, apo-NTD, or apo-HCP (from clade 4) 25,26 .…”

“…Both CTD and CTDH were shown to mediate carotenoid transfer to apo-OCP, apo-NTD, or apo-HCP (from clade 4) 25,26 . OCP and CTDH are also capable of taking up a carotenoid molecule from membranes while HCP4 and isolated NTD are unable to do so 25 .…”