Structural insight into the mechanism of energy transfer in cyanobacterial phycobilisomes

ARTICLE https://doi.org/10.1038/s41467-021-25813-y OPEN Structural insight into the mechanism of energy transfer in cyanobacterial phycobilisomes 1234567890():,; Lvqin Zheng1,5, Zhenggao Zheng2,3,5, Xiying Li2,5, Guopeng Wang1, Kun Zhang Jindong Zhao 2,4 ✉ & Ning Gao 1 ✉ 2, Peijun Wei2, Phycobilisomes (PBS) are the major light-harvesting machineries for photosynthesis in cyanobacteria and red algae and they have a hierarchical structure of a core and peripheral rods, with both consisting of phycobiliproteins and linker proteins. Here we report the cryoEM structures of PBS from two cyanobacterial species, Anabaena 7120 and Synechococcus 7002. Both PBS are hemidiscoidal in shape and share a common triangular core structure. While the Anabaena PBS has two additional hexamers in the core linked by the 4th linker domain of ApcE (LCM). The PBS structures predict that, compared with the PBS from red algae, the cyanobacterial PBS could have more direct routes for energy transfer to ApcD. Structure-based systematic mutagenesis analysis of the chromophore environment of ApcD and ApcF subunits reveals that aromatic residues are critical to excitation energy transfer (EET). The structures also suggest that the linker protein could actively participate in the process of EET in both rods and the cores. These results provide insights into the organization of chromophores and the mechanisms of EET within cyanobacterial PBS. 1 State Key Laboratory of Membrane Biology, National Biomedical Imaging Center, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, 100871 Beijing, China. 2 State Key Laboratory of Protein and Plant Genetic Engineering, School of Life Sciences, Peking University, 100871 Beijing, China. 3 College of Life Science, Qingdao University, 266071 Qingdao, China. 4 Key Laboratory of Phycology of CAS, Institute of Hydrobiology, Chinese Academy of Sciences, 430072 Wuhan, Hubei, China. 5These authors contributed equally: Lvqin Zheng, Zhenggao Zheng, Xiying Li. ✉email: ; NATURE COMMUNICATIONS | (2021)12:5497 | https://doi.org/10.1038/s41467-021-25813-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25813-y T he cyanobacteria were responsible for the rise of oxygen on earth about 2.4 billion years ago and they are one of the most important groups of organisms in carbon and nitrogen cycles in the biosphere1–3. The major light-harvesting system for solar energy capture in cyanobacteria and red algae is phycobilisome (PBS)4–8, which consists of phycobiliproteins (PBP) with covalently attached open-chain tetrapyrroles as chromophores (bilins) and linker proteins5,9,10. The basic unit of the PBS is a heterodimer of an α subunit and a β subunit (often called αβ monomer). Three αβ monomers form a ring-shaped trimer, and a hexamer is formed by a face-to-face stacking of two trimers. The hexamers are organized further into a highly ordered supramolecular complex of PBS with a nearly unity excitation energy transfer (EET) efficiency. Two recently determined cryo-EM structures of PBS from red algae11,12 revealed how the linker proteins organize the PBP into an ordered hierarchical architecture. In general, PBS found in nature is composed of two parts, a central core and peripheral rods that are attached to the core. With a few exception13, most cyanobacterial PBS have a hemidiscoidal shape and their peripheral rods are variable in length5. At present, the cyanobacterial PBS structure of high resolution is unavailable. To gain insights into the mechanism of EET in cyanobacterial PBS, we determined the cryo-EM structures of two cyanobacterial PBS, one with a tricylindrical core from Synechococcus sp. PCC 7002 (Synechococcus 7002) and the other with a penta-cylindrical core14,15 from Anabaena sp. PCC 7120 (Anabaena 7120). Results Overall structures of the cyanobacterial PBS. PBS complexes were purified from the cultures of Synechococcus 7002 and Anabaena 7120 following standard procedures15, and the samples were analyzed biochemically and spectroscopically for their compositional and functional integrity (Supplementary Fig. 1). Both PBS contain phycocyanin (PC), allophycocyanin (APC), and linker proteins but lack phycoerythrin in red algal PBS. Negativestaining electron microscopy (nsEM) analysis revealed a large variation in the length of peripheral rods (Supplementary Fig. 2a, b), and the longest rods could contain as many as six PC hexamers. Initial cryo-EM visualization indicated that the rods are unstable and the distal rod hexamers could readily dissociate from the PBS during cryo-grid preparation. To minimize the PBS complex disassembly, the samples were mildly cross-linked with a low concentration of glutaraldehyde (0.0125% and 0.005% for the Synechococcus 7002 and Anabaena 7120 PBS samples, respectively) before cryo-freezing. Because the purification buffer contained a high concentration of salt (0.75 M K + /Na + ) required for PBS stabilization, which produced undesired high background noise during cryo-EM imaging, an instant on-grid dilution of samples with a salt-free buffer was applied during grid preparation. With these optimizations of sample preparation, we determined the overall structures of PBS from Synechococcus 7002 and Anabaena 7120 at resolutions of 3.5 and 3.9 Å, respectively (Supplementary Figs. 3 and 4). As shown in Fig. 1, both PBS are hemidiscoidal in a twofold symmetry (Fig. 1a, e) with the cores located at the center and the peripheral rods attached to the cores. In addition to the rod length variation, as shown in typical 2D average images of side views (Supplementary Figs. 3c and 4c), the relative orientation of the rods to the core is also variable and the densities of the distal rod trimers become highly fragmented in the final cryo-EM maps. Therefore, we conducted our analysis of cyanobacterial PBS structures with rod length within two hexamers (Fig. 1a, b, e, f). For Synechococcus 7002 PBS, we modeled 36 APC αβ monomers in the core, 72 PC αβ monomers in the rods, 6 rod 2 linker proteins (LR), 6 rod-core linker proteins (LRC), and 6 core linker proteins (ApcC, formerly LC) in the map. The dimensions of the PBS from Synechococcus 7002 are ~450 Å in length, ~ 300 Å in height, and ~220 Å in thickness (Fig. 1a, b). There are six peripheral rods (rods R1/R1’, R2/R2’, and R3/R3’) and each rod contains two hexamers in our model (Fig. 1a, b). The central core contains three cylinders arranged as an equilateral triangle: cylinders A and A’ at the bottom and cylinder B on the top (Fig. 1a, b). All three cylinders are each composed of two hexamers (four trimers) joined together in a back-to-back fashion. For Anabaena 7120 PBS, we modeled 48 APC αβ monomers in the core, 84 PC αβ monomers in the rods, 6 rod linker proteins (LR), 8 rod-core linker proteins (three different LRC), and 8 core linker proteins (ApcC). The dimensions of Anabaena 7120 PBS are ~540 Å in leng (...truncated)