Structural Characterization of Carotenoid Binding Proteins in Cyanobacteria and Elucidation of their Role in Carotenoid Uptake and Transfer Mechanism for Photoprotection

Photodamage to the photosynthetic apparatus by excessive light radiation has led to the evolution of a variety of energy dissipation mechanisms. One mechanism, that exists in some cyanobacterial species, enables non-photochemical quenching (NPQ) of excitation energy within the phycobilisome (PBS) antenna complex by the Orange Carotenoid Protein (OCP) or homologs. The OCP contains the functional N-terminal domain (NTD) and regulatory C-terminal domain (CTD). Some cyanobacteria also have genes encoding for homologs to both the CTD (referred to as CTDH) and the NTD (referred to as helical carotenoid proteins, HCPs). The CTDH facilitates the uptake of carotenoids from the thylakoid membranes to be transferred to the HCPs. Holo-HCPs exhibit diverse functionalities such as carotenoid carriers, singlet oxygen quenchers, and in the case of HCP4, constitutive OCP-like energy quenching. Here, we present the first crystal structures of a holo-HCP4 which binds a canthaxanthin (CAN) molecule, at a resolution of 1.9 Å and of apo-HCP4 at a resolution of 3.1 Å, both from Anabaena sp. PCC 7120 ​[1][2]​. Based on these structures coupled with biochemical and computational methods, we propose models of the binding of the HCP4 to the PBS and the associated energy-quenching mechanism. Structural analysis reveals significant differences between the apo and holo-HCP4 forms. The differences between the two structures helped identify critical changes required for functionality. Computational analysis could then predict the dynamics of these regions of the protein. These changes, coupled with the potential dynamics of CTDH residues previously predicted ​[3][4]​ create a favorable environment for efficient ligand transfer. The transition between apo- to holo- forms, along with their interactions with CTDH, underscores the intricate molecular mechanisms underlying carotenoid uptake and energy quenching in cyanobacteria. These insights advance our understanding of protein-ligand interactions in photosynthetic systems, providing a foundation for further exploration of photoprotection strategies and potential applications in bioengineering and cargo delivery research.

References:

​​[1]          J. Sklyar, A. Wilson, D. Kirilovsky, and N. Adir, “Insights into energy quenching mechanisms and carotenoid uptake by orange carotenoid protein homologs: HCP4 and CTDH,” Int J Biol Macromol, vol. 265, 2024, doi: 10.1016/j.ijbiomac.2024.131028.

​[2]          J. Sklyar, F. Glaser and N. Adir “The Apo-HCP4 Structure Reveals Facets of Carotenoid Uptake”, Submitted, 2025.

​[3]          D. Harris, F. Muzzopappa, F. Glaser, A. Wilson, D. Kirilovsky, and N. Adir, “Structural dynamics in the C terminal domain homolog of orange carotenoid protein reveals residues critical for carotenoid uptake,” Biochim Biophys Acta Bioenerg, vol. 1861, no. 8, p. 148214, 2020, doi:10.1016/j.bbabio.2020.148214.

​[4]          D. Harris, A. Wilson, F. Muzzopappa, N. Sluchanko, T. Friedrich, E.G. Maksimov, D. Kirilovsky, N.Adir, “Structural rearrangements in the C-terminal domain homolog of Orange Carotenoid Protein are crucial for carotenoid transfer,” Commun Biol, vol. 1, no. 1, 2018, doi: 10.1038/s42003-018-0132-5.

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