Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP

Maria Agustina Dominguez-Martin,Daniel J Rosenberg,Michal Hammel,Corie Y Ralston,Christopher J Petzold,Tomáš Polívka,Sayan Gupta,Cheryl A Kerfeld,Markus Sutter,Sigal Lechno-Yossef,Yan Chen

doi:10.1038/s41598-020-72383-y

Abstract

The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria. The 35 kDa OCP is structurally and functionally modular, consisting of an N-terminal effector domain (NTD) and a C-terminal regulatory domain (CTD); a carotenoid spans the two domains. The CTD is a member of the ubiquitous Nuclear Transport Factor-2 (NTF2) superfamily (pfam02136). With the increasing availability of cyanobacterial genomes, bioinformatic analysis has revealed the existence of a new family of proteins, homologs to the CTD, the C-terminal domain-like carotenoid proteins (CCPs). Here we purify holo-CCP2 directly from cyanobacteria and establish that it natively binds canthaxanthin (CAN). We use small-angle X-ray scattering (SAXS) to characterize the structure of this carotenoprotein in two distinct oligomeric states. A single carotenoid molecule spans the two CCPs in the dimer. Our analysis with X-ray footprinting-mass spectrometry (XFMS) identifies critical residues for carotenoid binding that likely contribute to the extreme red shift (ca. 80 nm) of the absorption maximum of the carotenoid bound by the CCP2 dimer and a further 10 nm shift in the tetramer form. These data provide the first structural description of carotenoid binding by a protein consisting of only an NTF2 domain.

Highlights

The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria
We used size exclusion chromatography (SEC)-small-angle X-ray scattering (SAXS) to characterize the oligomerization states and X-ray footprinting-mass spectrometry (XFMS) to identify carotenoidbinding residues of the CCP2 isolated from its native host, Tolypothrix
We find that Tolypothrix CCP2 is predominantly a dimer (Fig. 3) with a single carotenoid molecule spanning the two monomers (Figs. 4A,D, 5A,B and S6)

Summary

Introduction

The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria. With the increasing availability of cyanobacterial genomes, bioinformatic analysis has revealed the existence of a new family of proteins, homologs to the CTD, the C-terminal domain-like carotenoid proteins (CCPs). 80 nm) of the absorption maximum of the carotenoid bound by the CCP2 dimer and a further 10 nm shift in the tetramer form These data provide the first structural description of carotenoid binding by a protein consisting of only an NTF2 domain. The first crystal structure of the OCP revealed two discrete structural domains: an all-helical N-terminal domain (NTD) unique to cyanobacteria, and a C-terminal domain (CTD) that is a member of the widespread NTF2-like superfamily (pfam02136). When recombinantly expressed in a canthaxanthin(CAN)-producing strain of E. coli, the CTD binds carotenoid[10,11] and were so named C-terminal domain-like carotenoid proteins (CCPs)[10]. HCPs have begun to be biochemically and structurally c haracterized[9,13,14,15], revealing that the HCPs are monomers that bind a single carotenoid[9,14]

Methods

Results

Conclusion