Use of protein cross-linking and radiolytic footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II

Abstract

Protein cross-linking and radiolytic footprinting coupled with highresolution mass spectrometry were used to examine the structure of PsbP and PsbQ when they are bound to Photosystem II. In its bound state, the N-terminal 15-amino-acid residue domain of PsbP, which is unresolved in current crystal structures, interacts with domains in the C terminus of the protein. These interactions may serve to stabilize the structure of the N terminus and may facilitate PsbP binding and function. These interactions place strong structural constraints on the organization of PsbP when associated with the Photosystem II complex. Additionally, amino acid residues in the structurally unresolved loop 3A domain of PsbP ( 90 K-107 V), 93 Y and 96 K, are in close proximity (≤11.4 Å) to the N-terminal 1 E residue of PsbQ. These findings are the first, to our knowledge, to identify a putative region of interaction between these two components. Cross-linked domains within PsbQ were also identified, indicating that two PsbQ molecules can interact in higher plants in a manner similar to that observed by Liu et al. [(2014) Proc Natl Acad Sci 111 (12):4638-4643] in cyanobacterial Photosystem II. This interaction is consistent with either intra-Photosystem II dimer or inter-Photosystem II dimer models in higher plants. Finally, OH • produced by synchrotron radiolysis of water was used to oxidatively modify surface residues on PsbP and PsbQ. Domains on the surface of both protein subunits were resistant to modification, indicating that they were shielded from water and appear to define buried regions that are in contact with other Photosystem II components.oxidoreductase that is found in all oxygenic photosynthetic organisms. This membrane protein complex contains at least 20 protein subunits, 17 of which are intrinsic membrane proteins. Higher plants contain three extrinsic proteins associated with the complex-PsbO, PsbP, and PsbQ-whereas cyanobacteria contain PsbO, PsbU, PsbV, and CyanoQ (a homolog of PsbQ). In higher plants the PsbO, PsbP, and PsbQ proteins are required for optimal rates of O 2 evolution under physiological inorganic calcium and chloride concentrations (1, 2).The PsbO protein appears to play a central role in the stabilization of the manganese cluster in all oxygenic photosynthetic organisms (3). In higher plants, PsbO and PsbP are required for photoautotrophic growth, PS II assembly, and the stabilization of PS II supercomplexes (4-9), with PsbP also being required for normal thylakoid assembly (6). Under low-light growth conditions, PsbQ is required for photoautotrophy (10).Although the crystal structure of cyanobacterial PS II has been resolved to 1.9 Å (11), no crystal structures for higher plant PS II have been presented. The structure and interactions of PsbO with the intrinsic subunits have been approximated by analogy to the cyanobacterial photosystem. Although high-resolution crystal structures of isolated spinach PsbP [1.98 Å; Protein Data Bank (PDB) ID code 2VU4] (12) and PsbQ (1.49 Å; PDB ID code 1VYK) are avai...

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga

“…Very recently, however, Liu et al (44) reported that cyano-PsbQ was located in the middle of the PSII dimer between two PsbO subunits from two monomers in the lumenal surface of cyanobacterial PSII and interacts with CP47 instead of CP43, based on the cross-linking of a PsbQ dimer and subsequent mass spectrometric analysis. A similar cross-linked dimer of PsbQ was observed with higher plant PSII, suggesting a similar arrangement of the PsbQ in the higher plant PSII (45). This is different from the site of PsbQЈ revealed by the present crystallographic studies, as well as the single particle image analysis of the red algal PSII (43).…”

“…Importantly, the interaction of PsbQ with CP43 has also been confirmed in higher plant PSII by cross-linking studies (46), which is consistent with the location of PsbQЈ we observed in the red algal PSII. In addition, Mummadisetti et al (45) suggested that the observation of cross- linked PsbQ dimers in higher plant PSII can also be explained by cross-linking of the anti-parallel PsbQ bridging two different adjacent PSII dimers without restraining the location of this subunit in the interface region of the two monomers. This is consistent with the location of this subunit revealed in the present study and may indicate a different location of PsbQ between prokaryotic and eukaryotic PSII.…”

The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes

“…However, the composition of these protrusions in grana membranes from plants and algae is not completely resolved. The precise locations of PsbP and PsbQ are still uncertain (Ido et al, 2014;Mummadisetti et al, 2014) because only cyanobacterial PSII-OEC crystal structures have been solved (Zouni et al, 2001;Kamiya and Shen, 2003;Ferreira et al, 2004;Loll et al, 2005;Umena et al, 2011;Kern et al, 2013Kern et al, , 2014Kupitz et al, 2014, Suga et al, 2015 and they do not have the PsbP and PsbQ proteins. PsbO is conserved among cyanobacteria, algae, and plants, and its position within PSII-OEC is more certain (Nield and Barber, 2006).…”

Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution

“…Curiously, the elongated N-terminal region of spinach PsbQ reaches out approximately 50 Å away from the four-helix bundle and binds to a long loop of PsbP between Lys90 and Ala111. The interaction between PsbP and PsbQ was previously detected through a crosslinking method combined with mass spectrometry 25,26 . Marked conformational changes occur in the flexible regions of PsbP and PsbQ when they bind to PSII core subunits (Extended Data Fig.…”