The Conserved His-144 in the PsbP Protein Is Important for the Interaction between the PsbP N-terminus and the Cyt b559 Subunit of Photosystem II

Abstract

Background:PsbP is an extrinsic subunit of photosystem II in green plants. Results: H144A mutation in PsbP alters the chloride requirement and affects the interaction between its N terminus and the PsbE component of photosystem II. Conclusion:The N-and C-terminal domains of PsbP cooperate to support PSII activity. Significance: This provides important information about the binding characteristics of PsbP in green plant PSII.The PsbP protein regulates the binding properties of Ca 2؉ and Cl ؊ , and stabilizes the Mn cluster of photosystem II (PSII); however, the binding site and topology in PSII have yet to be clarified. Here we report that the structure around His-144 and Asp-165 in PsbP, which is suggested to be a metal binding site, has a crucial role for the functional interaction between PsbP and PSII. The mutated PsbP-H144A protein exhibits reduced ability to retain Cl ؊ anions in PSII, whereas the D165V mutation does not affect PsbP function. Interestingly, H144A/D165V double mutation suppresses the effect of H144A mutation, suggesting that these residues have a role other than metal binding. FTIR difference spectroscopy suggests that H144A/D165V restores proper interaction with PSII and induces the conformational change around the Mn cluster during the S 1 /S 2 transition. Cross-linking experiments show that the H144A mutation affects the direct interaction between PsbP and the Cyt b 559 ␣ subunit of PSII (the PsbE protein). However, this interaction is restored in the H144A/D165V mutant. In the PsbP structure, His-144 and Asp-165 form a salt bridge. H144A mutation is likely to disrupt this bridge and liberate Asp-165, inhibiting the proper PsbP-PSII interaction. Finally, mass spectrometric analysis has identified the cross-linked sites of PsbP and PsbE as Ala-1 and Glu-57, respectively. Therefore His-144, in the C-terminal domain of PsbP, plays a crucial role in maintaining proper N terminus interaction. These data provide important information about the binding characteristics of PsbP in green plant PSII.Photosystem II (PSII) 2 consists of both membrane-intrinsic and membrane-extrinsic subunits, and functions as a water/ plastoquinone oxidoreductase (for reviews, Refs. 1-4). On the thylakoid lumenal side of PSII, a metal cluster of four Mn ions, one Ca 2ϩ ion, and five oxo ligands (the Mn cluster) catalyzes the oxygen-evolving reaction. Additionally, two Cl Ϫ ions are bound near to the Mn cluster (5). The membrane-intrinsic subunits of PSII are involved in pigment and/or cofactor binding for photochemical reactions, while the membrane-extrinsic subunits surround the catalytic Mn cluster and play crucial roles in stabilizing the Mn cluster and retaining the PSII cofactor ions (6 -8).X-ray structural analysis of the cyanobacterial PSII complex at atomic resolution has revealed the location of subunits, pigments, and cofactors, including the exact organization of the subunits within PSII (5, 9 -11). However, the crystallographic information gained from the study of cyanobacterial PSII cannot necessarily be...

Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

“…3B shows the spectrum of ESSTPVVDGKQYY 127 and FKGAKKF 175 from PsbP and GANCGPEAVW 99 from CP26, in which the Glu-96 in CP26 and the Lys-174 in PsbP were cross-linked. The cross-linking between Ala-1 in PsbP and Glu-57 in PsbE was also detected in P-4, as reported previously (22). The scores of those peptides were statistically significant (p Ͻ 0.001, supplemental Tables S7 and S8), and the amino acid residues identified as cross-linked sites are highly conserved in their respective proteins in higher plants.…”

“…Plasmid Construction, Recombinant Protein Expression, and Purification-The recombinant PsbP_A186C, in which Ala-186 is substituted with Cys, was produced as reported previously (22). To produce the expression plasmid for PsbQ_ G149C, in which Gly-149 is substituted with Cys, cDNA fragments of spinach PsbQ were amplified by PCR with the primers (5Ј-CATATGAGGCCAGGCCCATCGTTGT-3Ј and 5Ј-GAGCTCTTAACAGAGCTTGGCAAGAAC-3Ј) using 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 30 s. The amplified fragment was purified, digested with NdeI and SacI, and subcloned into the pET 41-a vector (Novagen).…”

“…The MS analyses indicated that the bands P-1 to P-4 contained CP43, CP26, PsbR, and PsbE in addition to PsbP, respectively (Table 1 and supplemental Tables S1-S4). The band P-4 corresponds to the cross-linked peptide between PsbP and PsbE that we reported recently (22). Molecular masses of P-1 to P-4 (about 60, 46, 33, and 30 kDa, respectively) indicate that those subunits were cross-linked in a one-to-one (1:1) manner.…”

Localization and functional characterization of the extrinsic subunits of photosystem II: an update

“…However, recent studies using chemical cross-linking combined with mass spectrometry suggest the multiple and direct interaction of PsbP and PsbQ with membrane-intrinsic PSII subunits. Using the zero-length cross-linker 1-ethyl-3-(3-diethylaminopropyl) carbodiimide (EDC), Ido et al 63,64) revealed the existence of direct interactions between PsbP and PsbE and between PsbP and PsbR (Fig. 3).…”

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

“…This indicates that few of the residues located on this face are exposed to the bulk solvent. Residue 1 A, which earlier had been identified as interacting with PsbE: 57 E (26,29), and residues 96 K and 93 Y, which we have identified as interacting with PsbQ: 1 E, are all unmodified. Interestingly, the three residues that have recently been identified by Nishimura et al (38) 38 K, 143 K, 166 K, 170 K, and 174 K) whose modification with N-succinimidyl propionate + EDC prevented the association of PsbP with PS II.…”