Abstract
A recently reported family of soluble cyanobacterial carotenoproteins, homologs of the C-terminal domain (CTDH) of the photoprotective Orange Carotenoid Protein, is suggested to mediate carotenoid transfer from the thylakoid membrane to the Helical Carotenoid Proteins, which are paralogs of the N-terminal domain of the OCP. Here we present the three-dimensional structure of a carotenoid-free CTDH variant from Anabaena (Nostoc) PCC 7120. This CTDH contains a cysteine residue at position 103. Two dimer-forming interfaces were identified, one stabilized by a disulfide bond between monomers and the second between each monomer’s β-sheets, both compatible with small-angle X-ray scattering data and likely representing intermediates of carotenoid transfer processes. The crystal structure revealed a major positional change of the C-terminal tail. Further mutational analysis revealed the importance of the C-terminal tail in both carotenoid uptake and delivery. These results have allowed us to suggest a detailed model for carotenoid transfer via these soluble proteins.
Highlights
A recently reported family of soluble cyanobacterial carotenoproteins, homologs of the Cterminal domain (CTDH) of the photoprotective Orange Carotenoid Protein, is suggested to mediate carotenoid transfer from the thylakoid membrane to the Helical Carotenoid Proteins, which are paralogs of the N-terminal domain of the OCP
The C-terminal domain (CTD) interacts with the fluorescence recovery protein, which supports the reorganization of OCPR to OCPO12–14
The CTD homologs (CTDH) gene in Anabaena is located adjacent to the HCP4 gene, it was suggested that HCP4 and CTDH are the progenitors of OCP19,23,24
Summary
The AnaCTDH can form a large homogeneous oligomer. Anabaena apo-CTDH was prepared for crystallization as described in Muzzopappa et al (2017)[25]. The calculated Matthews coefficient indicated that the asymmetric unit could contain 10–24 AnaCTDH monomers Whether this large oligomerization state has any biological significance as a quaternary assembly has to be further investigated. The presence of high MW oligomers was detected in carotenoid-containing versions of AnaCTDH, TeCTDH, and Synechocystis CTD-OCP preparations obtained in Escherichia coli cells[25], suggesting that oligomerization is a more general characteristic of CTD-like proteins. These large oligomers were not detected in size exclusion chromatography when the holo-proteins were isolated from Synechocystis cells or when AnaCTDH was isolated under reducing conditions or in a AnaCTDH Cys103Phe mutant[25]. Resolution (Å) No of reflections Rwork/Rfree Completeness (%) No of non-hydrogen atoms
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