Early events of photosynthesis operate through a series of pigment protein complexes (PPSs). They harvest light energy and convert it to chemical energy. Many large multi-subunit complexes are partly understood, such as the reaction centers where photochemistry takes place, light-harvesting antenna complexes (LHCs) that increase the light energy capture cross-section [1]. However, it remains much less clear how these complexes interact with each other and how the excitation energy transfer is regulated [2]. Cyanobacterial light-harvesting complexes (Phycobilisome or PBS) are located on the stromal side of the thylakoid membrane. They are giant molecules, with a molecular mass range of 5-15 MDa 3, 4, 5. Light energy captured by PBS is usually transferred to Chlorophyll a (Chla) molecules in the photosystems (Photosystem I or PSI, and Photosystem II or PSII) where charge separation occurs. PBSs are comprised of highly organized assemblies of pigmented phycobiliproteins and colorless linker polypeptides 6, 7.

The pigment-free linker proteins play essential roles in PBSs’ structure and function. They fine-tune the chromophore spectral properties in favor of efficient excitation energy transfer from PBS to PSII and PSI 3, 5, 6, 7, 8. Many of these linker proteins contain a large portion of the unstructured loops or intrinsically disordered proteins/regions (IDPRs), a concept in contrast to the specific 3D structure-function paradigm 9, 10, 11. IDPRs of the linker proteins are often found at the interfaces between the protein subunits in PBS, such as the rod-core region, and the core-membrane anchoring region 3, 7, 12. ApcE, also termed as core-membrane linker or LCM [12], comprises both structured and unstructured domains (IDPRs), such as the structured phycocyanobilin binding domain (or PB domain or αLCM) and several linker domains (LDs) that are essentially required for connecting APC trimers (discs) 13, 14. Between the PB and LDs, there are several unstructured loops (IDPRs) connecting PB domain and LDs. Interestingly, a peptide loop (PB-loop), a 60-70 amino acid long, flexible IDPR, is even inserted into the structured PB domain [15]. This naturally occurred insertion does not interrupt the function of PB as a terminal energy emitter (TEE) through which the excitation energy is transmitted from PBS to the RCs. Genetic deletion of the PB-loop, however, suggested an altered energetic connectivity between PBS and the RCs, indicating its dynamic regulatory roles [16]. Note, the PB domain structure containing the intact PB-loop has not been resolved in either X-ray crystallography [17] or cryo-EM 3, 4, 5, 18 .

In this communication, several structural models of the PB domain were generated using the Google DeepMind’s AlphaFold protein 3D prediction algorithm. Experimentally, the isotopically-encoded chemical cross-linking technology in combination with the LC-MS/MS interrogations were applied in the current study. The goal is to elucidate the structural organization of the flexible PB-loop (IDPR) in the PB domain and in the context of whole PBS protein assembly. This work uses protein sequences of PBS from a cyanobacterium, Synechocystis sp. PCC 6803, (hereafter Synechocystis 6803). It was found that large portion of the PB-loop is located in the interface between disc 2 and disc 3 in each PBS basal cylinder. It is associated with the C-terminal helical domain of ApcG. The PB-loop and ApcG seem collectively form a protrusion towards the thylakoid membrane where the RCs are imbedded. A stepwise PBS assembly pathway, highlighting the structural roles of PB-loop and ApcG, is also proposed.