Orange carotenoid protein burrows into the phycobilisome to provide photoprotection

Abstract

In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS), the cyanobacterial antenna, induced by the orange carotenoid protein (OCP). A single photoactivated OCP bound to the core of the PBS affords almost total energy dissipation. The precise mechanism of OCP energy dissipation is yet to be fully determined, and one question is how the carotenoid can approach any core phycocyanobilin chromophore at a distance that can promote efficient energy quenching. We have performed intersubunit cross-linking using glutaraldehyde of the OCP and PBS followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) to identify cross-linked residues. The only residues of the OCP that cross-link with the PBS are situated in the linker region, between the N- and C-terminal domains and a single C-terminal residue. These links have enabled us to construct a model of the site of OCP binding that differs from previous models. We suggest that the N-terminal domain of the OCP burrows tightly into the PBS while leaving the OCP C-terminal domain on the exterior of the complex. Further analysis shows that the position of the small core linker protein ApcC is shifted within the cylinder cavity, serving to stabilize the interaction between the OCP and the PBS. This is confirmed by a ΔApcC mutant. Penetration of the N-terminal domain can bring the OCP carotenoid to within 5-10 Å of core chromophores; however, alteration of the core structure may be the actual source of energy dissipation.

Keywords: cross-linking; cyanobacteria; light harvesting; nonphotochemical quenching; photosynthesis.

Fig. 1.

Partial model of the Syn PBS. (A) The PBS is visualized from the direction of the thylakoid membrane. The model includes a single-basal core cylinder (including ApcA, ApcB, ApcC, ApcD, ApcE, and ApcF; colored in yellow, blue, light orange, gray, light green, and light blue, respectively) and a three-hexamer rod that includes the phycobiliproteins CpcA and CpcB (colored in pink and red, respectively) and the CpcC, CpcD, and CpcG linkers (in black). In all schematic models, linkers are shown as small objects because their position and structures are not known. Only the phycobiliprotein-type domain of ApcE is shown. (B) The same segment of the PBS is shown perpendicular to that in A (along the membrane). Colors are as in A.

Fig. S1.

Model of the Syn PBS. (A) The Syn PBS is visualized from the side, above the thylakoid membrane, similar to Fig. 1A but complete. An additional core cylinder and four rods are not seen from this angle. (B) Same model, seen along the membrane as in Fig. 1B.

Fig. S2.

Fluorescence spectra of different PBP–OCP complexes. PBS–OCP fluorescence spectra were produced using an excitation wavelength of 530 nm. Fluorescence of the preactivated fraction (dashed line) was normalized to 100%. Following OCP activation and PBS–OCP isolation by sucrose gradient centrifugation (dotted line), the fluorescence is almost completely quenched.

Fig. S3.

(A) SDS/PAGE of OCP–PBS cross-linked complexes. Lanes 1 and 2 show the isolated OCP and PBS, respectively. Lanes 3–6 show the results of exposure of the PBS–OCP complex for 2, 5, 10, or 15 min to 0.05% GA. The reactions were quenched and diluted into low-ionic-strength buffer to disassemble non–cross-linked complexes. (B) Fluorescence analysis (λ_ex 530 nm with ±2.5-nm slit width) of the cross-linked samples showing increased quenching, due to cross-linking–induced stability of the complex in low-ionic-strength buffer. The sample cross-linked for 5 min was chosen for MS/MS analysis. CL, cross-linking.

Fig. 2.

Cross-link constraint-dependent model of the OCP^R–PBS complex. The OCP^R model was constructed of the Nterm (PDB ID code 4XB4; residues 20–165; red cartoon) and Cterm (based on PDB ID code 3MG1; residues 186–311; orange cartoon); the flexible domain was built using Phyre2 (residues 160–196; black). Half of a basal core cylinder was constructed from two APC trimers, associated according to Chang et al. (23). ApcA, ApcB, ApcD, ApcE, and ApcF subunits are depicted in yellow, blue, gray, wheat, and light blue surfaces, respectively. The ApcC subunit (magenta cartoon) was docked into the terminal trimer using the 1B33 structure and the alignment algorithm implemented in PyMOL. ApcB and OCP residues that participate in cross-linking are depicted in blue or red–green–blue (RGB)-colored sticks, respectively. Phycocyanobilin chromophores are depicted in Corey–Pauling–Koltun (CPK)-colored sticks and surrounded by yellow ovals. The hECN carotenoid molecule of the OCP [Nterm(hECN)] is shown as red sticks. Cross-links are shown as black lines. (A) The Cterm is anchored by the link between OCP(K249) and ApcB(K113). An additional link is formed by OCP(K185) at the end of the flexible loop and ApcB′(K26) on the adjacent monomer. (B) The flexible loop (black) crosses over the terminal trimer through the gap (highlighted by the black box) between ApcA subunits (yellow surface). The positions of the two zones of cross-linked residues on ApcB are signified by the black oval (K26/K28/R39) and triangle (R67). These residues are present in triplicate in the trimer. The Nterm is shown overlapping the trimers. This indicates that the terminal trimer must move away from the second trimer (Fig. 3). (C) Cross-links between flexible-loop residues R67/R70/R71/R185 and ApcB residues K26/K28/K39/K67. The OCP(R185) residue seen in A is shown to cross-link to ApcB′ (26), the B subunit of the adjacent monomer.

Fig. 3.

Schematic model of the PBS–OCP complex. The model shows a single core cylinder composed of four trimers. The bottom two trimers have been separated by the intercalation of the OCP Nterm (dark red), connected by the flexible loop (black line) to the Cterm (dark orange) that is suspended outside the cylinder. Due to the change in the cylinder structure, ApcC (light orange) moves out of the terminal trimer and into the space near the second trimer, interacting with the Nterm and enabling cross-linking to ApcF (light blue). A single rod is shown (CpcA and CpcB in pink and red, respectively), with the CpcG linker protein (black) jutting out toward the cylinder. The end of the flexible loop closest to the Nterm must be within 30 Å of residues of CpcG (whose structure is unknown), denoted in Table 1.

Fig. S4.

Movement of ApcC due to OCP binding. The terminal trimer (viewed from within the core cylinder) is shown in semitransparent surface, colored as in Fig. 2. The ApcC’s (magenta cartoon) original position was obtained by superimposing the trimer with the 1B33 structure. Following Nterm (red cartoon) binding, ApcC moves into the cylinder space (toward the viewer, denoted as ApcC′ in pink) in a fashion that allows cross-linking to ApcF (black line).

Fig. 4.

ApcC depletion weakens the interaction between OCP and PBSs in vitro. Kinetics of fluorescence decrease in the WT (red) and ΔApcC (blue) PBSs (A and B) in 0.5 M (A) and 0.8 M phosphate buffer (B), and CK and CK-ΔApcC (C and D) in 0.8 M (C) and 1.4 M phosphate (D) were recorded with a PAM fluorometer. Isolated PBSs (0.012 μM) were used for each measurement. Different OCP concentrations were used, giving OCP-to-PBS ratios of 40 (squares), 20 (triangles), and 8 (circles). OCP was added in its dark, inactive orange form (OCP^O). The actinic light source, providing 900 μmol photon⋅m⁻²⋅s⁻¹ blue-green light and allowing conversion to the active red form (OCP^R), was turned on at t 0. Three independent experiments were performed, and the error bars (SE) are smaller than the symbols.

Fig. S5.

Fluorescence spectra of different PBSs. WT (red) and ΔApcC (blue) (A) and CK (red) and CK-ΔApcC (blue) (B) room temperature fluorescence spectra. Fluorescence emission was obtained by excitation at 590 nm.

Fig. S6.

Calculated surface electrostatic potential complementarity suggests the possible Nterm binding niche. The surface electrostatic potential of the components of the core–ACP model were calculated using the PyMOL vacuum algorithm (35). Negative and positive potentials (red and blue, respectively) are depicted on the same scale. (A) The ApcC subunit, based on the 1B33 structure (24), is mostly positively charged. (B) The Nterm (or RCP) from the 4XB4 structure (20) has a long positive patch along its entire length (black oval), parallel to the carotenoid chromophore (not shown). Other surfaces are mixed positive and negative. (C) Model of the Nterm bound to the APC trimer. The trimer is shown edge-on, whereas the second APC trimer is shown schematically in light blue. The internal surface of the APC trimer aperture exhibits a niche that is similar in size to the Nterm (black oval). Movement of ApcC out of its position removes a partial occlusion of this niche. The Nterm and APC potentials have a high degree of complementarity. The position of the Nterm in this orientation will allow the linker residues to cross over to the outside of the core as shown in Fig. 2.