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

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.

Figure 1

Characterization of CCP2 purified from Tolypothrix by denaturing gel electrophoresis. Coomassie blue-stained SDS-PAGE and anti-his immunodetection of holo-CCP2 (left) and apo-CCP2 (right).

Figure 2

UV–Vis absorption spectra. (A) Absorption spectrum of CCP2 (red line), CAN-OCP1 from Tolypothrix (blue line, taken from) and CAN in THF (black line). The color of CAN dissolved in organic solvent (1) and the purified protein in solution (2) are shown in the inset. (B) Absorption spectrum of CCP2 after SEC. The fractions correspond to the dimer peak (pink line) and the tetramer peak (purple line).

Figure 3

Oligomeric state of holo- and apo-CCP2. Apo- (A) and holo- (B) CCP2 oligomerization states analyzed by analytical SEC with a Superdex 75 column. Protein molecular weight standards were used to estimate the molecular mass for each species (inset graph A).

Figure 4

Global structural analysis by SEC-SAXS-MALS. (A) Top: SEC-MALS-SAXS chromatograms for apo-CCP2 dimer (5 mg/mL, apo1), apo-CCP2 tetramer (10 mg/mL, apo2), holo-CCP2 (5 mg/mL holo). Solid lines represent the UV 280 nm (light blue) or integrated SAXS signal (dark blue) in arbitrary units, while symbols represent molecular mass (cyan) and Rg values for each collected SAXS frame (dark blue) versus elution time. (B,C) p(r) functions calculated for the experimental data (black) of apo-CCP2 dimer, trimer, and, tetramer; and holo-CCP2 dimer and tetramer, shown in Figure S4. The area of the p(r) is normalized relative to the MW estimated by SAXS and is listed in Table S1. Theoretical p(r) function is calculated from the theoretical SAXS curves shown in Figure S4 of the corresponding models shown in (D,E). (D) Comparison of the experimental p(r) functions of holo-CCP2 dimer (blue) and apo-CCP2 dimer (red) together with the derived 3D structural models (see Materials and methods). C-terminal region (C-term) is highlighted. (E) Comparison of the experimental p(r) functions of holo-CCP2 tetramer (magenta) and apo-CCP2 dimer (green) together with the derived 3D structural models (see Materials and methods). The position of the conserved cysteine is highlighted in yellow.

Figure 5

Analysis of the conserved cysteine in CCP2. Position of the cysteine in the apo- (A) and holo- (B) structural models of CCP2 dimer. (C) Absorption spectrum of the holo-CCP2 with different concentrations of DTT. (D) Analysis of the presence of the disulfide bond by SDS-PAGE with and without reductant. Numbers on the left correspond to the molecular weight marker (lane 1) in kDa. (E) P(r) functions obtained for the SAXS profile of apo-CCP2 measured in solution with (5 mM TCEP) (green line) and without reductant (red line) in comparison to the holo-CCP dimer (blue line). (F) P(r) functions obtained for the SAXS profile of holo-CCP2 measured in solution with (green line) and without reductant (blue line) in comparison to the apo-CCP dimer (red line). Experimental SAXS profiles used to calculate P(r) functions are shown in the Figure S4.

Figure 6

XFMS data for carotenoid-binding residues within the CCP2 dimer. (A) Hydroxyl radical reactivity rates for the peptides implicating carotenoid-binding residues. (B) The identified carotenoid-binding residues mapped onto a monomer of a holo-dimer CCP2.