Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates

Abstract

Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn 4 Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27ΔctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3ΔctpAΔpsb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27ΔctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as

The Psb27 Assembly Factor Binds to the CP43 Complex of Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803

“…It therefore seems likely that Psb27 is bound to CP43 in the vicinity of this loop region and that binding might prevent access of a protease to the loop. Such a location is in agreement with the recent detection of a cross-link between CP43-Asp-321 and Psb27-Lys-63 (Liu et al, 2011a). Earlier in silico docking models had proposed binding sites for Psb27 close to that of the bulk of PsbO (Nowaczyk et al, 2010;Fagerlund and Eaton-Rye, 2011).…”

“…5). In addition, parallel work has shown that Psb27 can be cross-linked to CP43 in isolated His-tagged Psb27-containing PSII core complexes (Liu et al, 2011a). It is important to mention that the binding of Psb27 to CP43 was more stabilized under conditions of BNthan CN-PAGE.…”

MS-based cross-linking analysis reveals the location of the PsbQ protein in cyanobacterial photosystem II

“…PSII preparations were resuspended at 0.2 mg/mL chlorophyll a in 25% (wt/vol) glycerol, 10 mM MgCl 2 , 5 mM CaCl 2 , and 50 mM MES buffer (pH 6.5). Cross-linking of PSII samples with EDC was performed essentially as described previously (52). DTSSP and BS 3 (Thermo Scientific) cross-linking was performed according to the manufacturer's protocol (with minor modifications) followed by desalting by using Zeba spin columns (Thermo Scientific).…”

Regulatory factors for the assembly of thylakoid membrane protein complexes

“…A second psb27 homologue in Arabidopsis is required for efficient repair of photodamaged PSII, but not for PSII accumulation [67]. However, in Synechocystis, Psb27 has been implicated in the assembly of the Mn 4 Ca cluster and the stabilization of unassembled CP43 [68][69][70].…”