J. Biochem. 2014;156(4):203–210

doi:10.1093/jb/mvu028

Identification of the roles of individual amino acid residues of the helix E of the major antenna of photosystem II (LHCII) by alanine scanning mutagenesis Received January 29, 2014; accepted April 1, 2014; published online April 21, 2014

Cheng Liu, Yan Rao, Lei Zhang and Chunhong Yang* Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R. China

The functions of the helix E (W97-F105), an amphiphilic lumenal 310 helix of the major antenna of photosystem II (LHCII), are still unidentified. To elucidate the roles of individual amino acid residue of the helix E, alanine scanning mutagenesis has been performed to mutate every residue of this domain to alanine. The influence of every alanine substitution on the structure and function of LHCII has been investigated biochemically and spectroscopically. The results show that all mutations have little impact on the pigment binding and configuration. However, many mutants presented decreased thermo- or photo-stability compared with the wild type, highlighting the significance of this helix to the stability of LHCII. The most critical residue for stability is W97. The mutant W97A yielded very fragile trimeric pigment protein complexes. The structural analysis revealed that the hydrogen bonding and aromatic interactions between W97, F195, F194 and a water molecule contributed greatly to the stability of LHCII. Moreover, Q103A and F105A have been identified to be able to reinforce the tendency of aggregation in vitro. The structural analysis suggested that the enhancement in aggregation formation for Q103A and F105A might be attributed to the changing hydrophobicity of the region. Keywords: photosynthesis/aggregation hydrophobicity/light-harvesting chlorophyll a/b complexes of photosystem II/photosynthesis/stability. Abbreviations: CD, circular dichroism; Chl, chlorophyll; CMC, critical micelle concentration; DM, dodecyl b-D-maltoside; LHCII, major light-harvesting chlorophyll a/b complexes of photosystem II; Lut, lutein; PAGE, polyacrylamide gel electrophoresis; Neo, neoxanthin; NPQ, non-photochemical quenching.

Plants absorb sunlight primarily through a series of pigment-binding membrane proteins called light-harvesting complexes (1, 2). The major lightharvesting complexes of photosystem II (LHCII) is

Materials and Methods Site-directed mutagenesis Site-directed mutagenesis to LHCII was carried out with MutanBEST site-directed mutagenesis Kit (Takara, Japan). The primers for mutagenesis are as follows: W97A, 50 - CTG CCT TGA ACG CCA CAG CTT-30 ; F98A, 50 - ATC CTG CCT TTG CCC ACA CAG C- 30 ; K99A, 50 -AGA TCC TGC TGC GAA CCA CAC AG-30 ; G101A, 50 -GAT TTG AAG TGC TGC CTT GAA

ß The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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*Chunhong Yang, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R. China. Tel: þ86-01062836252, Fax: þ86-01062836219, email: [email protected]

the most abundant membrane protein in the world and binds almost half of the pigments in the thylakoid membrane (3). The basic unit of LHCII in vivo is trimer, which can form aggregations under different stimuli (4—6). The three-dimensional crystal structures of LHCII from spinach or from pea at 2.72 or 2.5 A˚ resolution, respectively, have been resolved (7, 8), which show that LHCII contains three transmembrane helices (B, C and A) and two amphiphilic helices (D and E). Helix E is a 310 helix located beside an antiparallel strands in the lumenal loop between helices B/C. LHCII binds eight chlorophyll (Chl) a and six Chl b, together with two luteins (Lut), one neoxanthin (Neo) and one violaxanthin that can be converted to zeaxanthin through xanthophyll cycle under overexcitation conditions (9, 10). LHCII is a multifunctional pigment—protein complex that performs structural and functional regulations under different light conditions. Controversial proposals have been made to explain the mechanisms regulating non-photochemical quenching (NPQ) of excessive energy by LHCII (11—14). One of them proposes that the subtle reversible adjustment of the configuration of pigments in LHCII, induced by protein conformation change, exists during the switching of functions (15), whereas the other one believes that LHCII does not undergo any conformational change by regulating different functions (8). LHCII is one of a few membrane proteins that can be reconstituted in vitro (16, 17); therefore, mutagenesis analysis can be used to study the roles of specific residues or domains of LHCII (18—25). Our previous study has discovered that the antiparallel strands in the lumenal loop between helices B/C of LHCII is important for configuration of Neo and the trends of aggregation in vitro (22, 25). In this study, alanine substitutions have been performed in the helix E (W97F105) to reveal the structural and functional roles of individual residue of the domain (except residue 100, which is originally an alanine). The results showed that the side chain of many residues, especially W97, in helix E contribute to the (thermo- and photo-) stability of the complexes and mutations at two residues (Q103 and F105) impact the aggregation formation of LHCII in vitro.

C. Liu et al. CCA C-30 ; S102A, 50 -GAT TTG AGC TCC TGC CTT GAA CCA C-30 ; Q103A, 50 -TCG CTA AAG ATA GCA GAT CCT GCC T-30 ; I104A, 50 -CCC TCG CTA AAT GCT TGA GAT CCT G-30 ; F105A, 50 -ACC CTC GCT GGC GAT TTG AGA T-30 . The apoproteins were overexpressed and isolated as the method described in Ref. 17. Preparation of recombinant LHCII Refolding of LHCII was executed following the method described in Refs. 16 and 17. The reconstitution mixtures were loaded onto sucrose density gradients and centrifuged for 18 h in Beckman SW55 Ti rotor at 300,000 g and 4 C. The bands corresponding to trimers were harvested for the structural and functional analysis. Partially denaturing polyacrylamide gel electrophoresis (PAGE) was carried out as described in Ref. 26.

Spectroscopic analysis The absorption spectra were recorded using Shimadzu UV-VIS 2550 spectrophotometer (Shimadzu, Japan) at room temperature. The wavelength step and the slit width were set to 0.5 and 1 nm, respectively. The 77 K fluorescence emission spectra at a range of 600—780 nm were recorded with a Hitachi F-4500 spectrofluorometer (Hitachi, Japan) with the excitation wavelength of 480 nm, and the slit is 2.5 nm. The circular dichroism (CD) spectra were recorded on a Jasco 815 spectropolarimeter (Jasco, Japan) at 10 C. The spectra were measured from 350 to 750 nm at a scan rate of 100 nm min1. Thermo- and photo-stabilities analysis The thermo- and photo-stabilities of different LHCII species were analysed as described in Ref. 22. Briefly, the thermal stability was measured by observing the decrease in the extent of energy transfer from complex-bound Chl b to Chl a upon gradual dissociation of the complexes at 37 C. The photo-stabilities were evaluated by the time course of the photobleaching of different LHCII species measured according to Ref. 22. The kinetics of themo- or photo-stabilities were fitted with approximate model, whose qualities were evaluated using the coefficient of determination R2. All R2 were 40.95. Characteristics of aggregation LHCII aggregation was induced by diluting the dodecyl b-D-maltoside (DM) concentration of the LHCII samples to 0.0025% (w/v), far below the critical micelle concentration (CMC). Then the samples were centrifuged at 20,000 g for 20 min at 4 C and resuspended again with the same buffer. Fluorescence emission spectra were measured at 77 K with the excitation wavelength 436 nm.

Results Reconstitution of different LHCII species and the pigment stoichiometries

Table I shows the pigment stoichiometries of all LHCII species. The wild type (WT) LHCII bound 6.6 Chl a, 5.5 Chl b and nearly 1 Neo per 2 Luts. All mutants except W97A possess similar pigment stoichiometries as the WT. W97A bound only half the amount of Chl b, compared with the WT, and 0.75 Neo per monomer. All the mutagenesis did not significantly influence capacities of protein folding into functional complexes (supplementary Figs. S1 and S2). W97A constructs very unstable pigment—protein complexes. 204

Absorption and fluorescence emission spectra, reflecting the transition energy and the energy transfer from Chl b to Chl a in LHCII, were measured at room temperature (Fig. 1A and C) and at 77 K, respectively (supplementary Fig. S3). The WT presented an absorption maximum of 674 nm in the Qy region, which was ascribed to Chl a, with one shoulder at 650 nm originated from Chl b. In the Soret region, two major peaks at 435 and 475 nm were observed. The second deviations of absorption spectra have been performed to reveal the differences in detail (Fig. 1B and D). All mutants except W97A possessed similar absorption peaks as the WT. W97A presented slightly reduced absorptions at 488 nm in the Soret region and 675 nm in the Qy region. The 77 K fluorescence emission spectra upon Chl b-excitation of different LHCII species shows clearly that none of the alanine substitutions of the helix E residues hindered the energy transfer from Chl b to Chl a. The mutation has changed the LHCII comformation

The CD spectra in the visible range reflect the conformation of the LHCII-bound pigments (Fig. 2). The WT shows typical CD spectrum of LHCII with two negative CD bands at 680 and 650 nm in the Qy region, and two negative CD bands at 473 and 493 nm in the Soret region. The ratio of the amplitudes of the two Soret bands at 474 and 493 nm (CD473/CD493) is 0.75. It is shown that some mutagenesis in the helix E changed the CD spectra individually. In particular, the peak at 650 nm of W97A is decreased, suggesting that the Chl b-involved interactions in LHCII were disturbed. Similar but smaller reductions also exist in mutants G101A, I104A and F105A. In the Soret region, the ratio CD473/CD493 of Q103A and F105A increased to 1. The mutants F98A, K99A and S102A present similar CD spectra as WT. The mutagenesis in the helix E reduced the thermal stability of LHCII

The thermal stabilities of all LHCII samples, reflected by the decay of energy transfer from Chl b to Chl a at 37 C (supplementary Fig. S4), were fitted to a two-phase exponential decay (Y ¼ Y0 þ A1*exp (x/T1)þA2*exp (—x/T2)) (Table II). The Y0 value indicates the proportion of the energy transfer protected from the treatment; the A1 and A2 values indicate the losses of the energy transfer in the two components, respectively;  1 and  2 are the half-life of the two components, respectively (¼ T*ln2). It is clear from Table II that all the mutants showed shorter lifetimes and less-protected Chls, revealing that all the mutagenesis of the helix E residues led to the reduction of functional stability, shown by the lower Y0 values and halftimes than those of WT. W97A presented the lowest thermal stability, whose Y0 value was only one-third of that of WT.

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Pigment analysis The pigments were extracted with 2-butanol according to the method described in Ref. 27. The 2-butanol extraction was further analysed for pigment stoichiometry with high-performance liquid chromatography as described in Ref. 26.

The mutation has not changed transition energy and energy transfer from Chl b to Chl a of the LHCII

Roles of individual amino acid residues of the helix E Table I. Pigment composition of the recombinant LHCII samples. Lhcb1

Chl a

Chl b

Lut

Neo

WT W97A F98A K99A G101A S102A Q103A I104A F105A

6.62  0.11 6.73  0.12 6.72  0.15 6.59  0.13 6.49  0.2 6.56  0.13 6.65  0.15 6.61  0.13 6.63  0.14

5.53  0.15 5.27  0.13 5.62  0.12 5.62  0.14 5.39  0.15 5.53  0.16 5.52  0.15 5.50  0.17 5.47  0.13

2 2 2 2 2 2 2 2 2

0.98  0.05 0.75  0.05 0.98  0.04 1.03  0.07 0.89  0.02 1.03  0.10 0.91  0.08 0.94  0.04 0.97  0.03

All values are normalized, assuming each LHCII monomer binds two Lut and are given as the average of 3—5 individual measurements  SD.

The mutagenesis in the helix E changed photo-stabilities of LHCII

The photobleaching kinetics of the different LHCII species subjected to strong illumination (Fig. 3A and B) could be fitted well with an exponential-linear decay (Y ¼ Y0 þ A*exp(x/p)k*x), suggesting that in our condition, the photobleaching of Chl in LHCII comprised of two phases, an exponential decay followed by a linear decay. Figure 3C presents three parameters (A, k and ) resulted from the fitting. A describes the amounts of Chls photobleached

occurred during the exponential process and k the velocity of the linear decay process. The parameter , calculated with p ( ¼ p*ln2), is the half value time of the exponential decay. It is obviou from Fig. 3C that all the mutants showed decreased resistance against the high light treatment, except S102A, which was more sustainable to high light treatment. W97A showed the most striking effect with only 40% of the initial absorbance after 30-min illumination, compared with the 80% of remaining absorption for WT. 205

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Fig. 1 Absorption spectra of WT and mutant LHCII measured at room temperature (A, C) and their second-deviation spectra (B, D). The spectra were normalized at red-most absorbance maximum. WT (solid line), W97A (dash line), F98A (dot line), K99A (dash-dot line) and G101A (dashdot-dot line) in (A) and (B). WT (solid line), SA102A (dash line), Q103A (dot line), I104A (dash-dot line) and F105A (dash-dot-dot line) in (C) and (D).

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The mutagenesis in the helix E changed the tendency to aggregate

LHCII aggregation shows a characteristic fluorescence emission maximum at 700 nm in a 77 K fluorescence emission spectrum (6). The degree of aggregation can be quantitatively assessed by the ratio of the amplitudes of the two emission peaks at 700 and 680 nm (F700/F680). Figure 4 shows the comparison of 77 K fluorescence emission spectra of different LHCII species dissolved at 0.0025% (w/v) DM concentration, which is much less than the CMC value of DM (0.009% (w/v) in water). W97A presented one blueshifted peak at 670 nm, close to the emission peak of free Chl a. We believed that the structure of W97A was completely destroyed according to its low stability. F700/F680 of all the samples except W97A are listed in the inset of Fig. 4, which clearly indicated that Q103A (F700/F680 ¼ 0.92) and F105A (0.97) aggregated stronger than WT (0.83), while no differences were observed from the other mutants.

Discussion LHCII is a multifunctional protein in vivo, with an inbuilt ability to switch its functions between harnessing solar energy under moderate light conditions and dissipating excessive excited energy under strong light

Table II. Parameters of the dual exponential decay functions describing the decrease in the energy transfer from Chl b to Chl a due to thermal denaturation. Lhcb1

s1

s2

Y1

Y2

Y0

WT W97A F98A K99A G101A S102A Q103A I104A F105A

50.9  0.1 16.6  0.3 39.2  0.2 46.7  0.3 48.9  0.3 44.2  0.5 48.8  0.2 47.2  0.3 44.1  0.3

681.3  12.4 332.2  7.4 649.1  10.1 491.3  13.5 488.6  8.5 400.4  5.3 582.7  7.3 445.4  5.4 423.8  4.5

0.16  0.01 0.44  0.01 0.14  0.01 0.17  0.01 0.15  0.01 0.13  0.01 0.16  0.01 0.12  0.01 0.14  0.01

0.15  0.01 0.68  0.01 0.17  0.01 0.11  0.01 0.21  0.01 0.15  0.01 0.21  0.01 0.21  0.01 0.17  0.01

0.71  0.02 0.28  0.01 0.69  0.02 0.72  0.01 0.63  0.02 0.66  0.01 0.63  0.02 0.67  0.01 0.66  0.01

206

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Fig. 2 CD spectra of WT and mutant LHCII measured at room temperature. The spectra were normalized to the same Chl concentration. The spectra are vertically shifted for clarity.

conditions (28, 29). However, its molecular mechanisms still need to be unveiled. Sunku et al. (30) applied magic-angle spinning nuclear magnetic resonance to analyse the effect of Arg on the functional regulation of LHCII between light-harvesting and energy dissipation, which led to the hypothesis that functional regulation of LHCII likely involved the loop structure, particularly the one between helices B/C (23). Recently, single residue mutagenesis in this region has been found to affect the LHCII’s sensitivity to low pH in initiating the functional switch (18). Some key residues in the antiparallel strands in the lumenal loop were found to be of great structural and functional significance as well (22, 25). The crystal structural illumination of LHCII shows that there is a 310 helix with nine residues named helix E in the lumenal loop between helices B/C (7), the functions of which are still unknown. In this work, the roles of every individual residue in the helix E of LHCII were studied by alanine scanning mutagenesis, a powerful tool to determine the contribution of every single residue in a functional domain (31). In present study, although all the mutants were capable of binding thylakoid pigments to form functional monomers and trimers, according to the sucrose density ultracentrifuge results (supplementary Fig. S1), W97A failed to survive the PAGE analysis, a harsher process (supplementary Fig. S2). Therefore, it was not surprising when W97A trimers presented very poor performance in both thermo- and photo-stability measurements (supplementary Fig. S4, Fig. 3 and Table II). Crystal structural data (7) reveal that W97 lies in the core of the complexes and the side chain is very close to those of F194 and F195 (Fig. 5A). The distance between the centres of the aromatic rings is likely in the range of 4—6 A˚ (the shortest distance between W97 and F194 or F195 are 4.5 A˚ and 3.5 A˚, respectively), allowing aromatic interaction, which is also favoured by the edge-to-face orientation of the residues (32). These interactions, together with those from two Luts, may contribute greatly to the LHCII stability (Fig. 5A). When W97 are mutated to alanine, the aromatic interactions are totally lost (Fig. 5B), which results in deterioration of the stability of LHCII. The stability analysis also indicated that many mutants also present reduced thermo- and photo-stability (supplementary Fig. S4, Fig. 3 and Table II), suggesting that the side chains of these

Roles of individual amino acid residues of the helix E

Fig. 3 Photobleaching of WT and mutant LHCII. The decay curves (A and B) show the total Qy absorption relative to the initial value (the initial value was set to 1). (C) shows parameters of the exponential-linear decay describing the decrease in the Chl absorption due to photobleaching.

residues all contribute to the stability of LHCII. Under moderate light condition, the lumenal pH is neutral or slightly basic. However, under strong light, the lumen will be acidified to about pH 5.5, or even 4.5 at the extreme condition (33, 34). The molecular dynamics simulation of the structure of the lumenal loop in WT LHCII and its E94G mutant showed that the mutagenesis of E94 to G resulted in a shorter 310 helix than that of WT LHCII at pH 5.5. Interestingly, this mutant in vitro presents a very different CD spectrum at pH 5.4 from that at pH 7.5, which suggests great pigment conformation discrepancy. As a control, WT LHCII maintains similar pigment conformation at pH 7.5 and pH 5.4 in vitro (25). In this study, it is evident that one single mutation in this region can also impact stability under heat or strong light condition, which can induce lumenal

acidification in vivo. Therefore, we proposed that the 310 helix might function as ‘stabilizer’ to the conformation of LHCII under stressful circumstances, which plants may encounter in vivo, such as heat or strong light condition. Helix E, located in the periphery of LHCII, is amphipathic. None of the residues in the helix E serves as a binding site of Chls (7). F98A, K99A, G101A, S102A and I104A presented almost similar pigment stoichiometries and spectroscopic characteristics to the WT, which suggests that these mutations have trivial impacts on the pigment binding and conformation. On the contrary, the W97A causes 0.26 Chl b and 0.23 Neo losses (Table I). Although the fluorescence emission spectrum, upon Chl b excitation, indicates that the energy transfer from Chl b to Chl a is intact in the W97A (supplementary Fig. S3), the pigment conformation is largely changed as shown by the absorption and CD spectra (Figs. 1 and 2). It was proposed that the low thylakoid lumenal pH induced by overexcitation led to aggregate formation of LHCII, in switching to the photoprotective state (12). The mechanisms for LHCII aggregation are still not settled. Aggregation of LHCII can be induced by low detergent concentration in vitro (6), which is suggested to resemble the in vivo NPQ upon LHCII 207

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Fig. 4 Seventy-seven kelvin fluorescence emission spectra of WT and mutant LHCII excited at 436 nm. WT (solid line), W97A (dash line), F98A (dot line), K99A (dash-dot line) and G101A (dash-dot-dot line) in (A). WT (solid line), SA102A (dash line), Q103A (dot line), I104A (dash-dot line) and F105A (dash-dot-dot line) in (B). Inset, the ratio of the amplitude of two emission peaks at 700 and 680 nm of WT and mutant LHCII.

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Fig. 5 Comparison of the molecular structures of helix E of WT and mutant LHCII. The figure illustrates the positions of W97 (A), Q103 (C) and F105 (E) in the WT and the suggested positions of introduced Ala in W97A (B), Q103A (D) and F105A (F) mutant, respectively. Water molecules are indicated by small spheres. Hydrogen bonds are represented by yellow dashed lines and the distances between them are indicated. The analysis was done with the software Pymol.

aggregate (12). In this study, the significantly higher F700/F680 ratios of Q103A (0.97) and F105A (0.93) than that of the WT (0.83) revealed stronger tendencies of the two mutants to form aggregation (Fig. 4). It can 208

be seen in the structure of LHCII (7) that F105 is located very close to W222 of another monomer and one digalactosyl diacyl-glycerol molecule (Fig. 5E). The aromatic interaction between F105 and W222

Roles of individual amino acid residues of the helix E

Conclusion We applied alanine scanning mutagenesis analysis to study the structural and functional roles of each residue in the helix E (a relatively long 310 helix) of LHCII. The results showed that, in general, mutations at most residues induced deteriorated thermo- or photo-stability, suggesting that the side chain of these residues contribute to the resistance of the complexes against high temperature and high light treatment. That W97A severely damaged the stability of the complexes highlights the importance of the aromatic interaction and hydrogen bonds of this residue in combination with the structural analysis. We also discovered that several mutants, such as Q103A and F105A, significantly enhanced the aggregate formation.

Supplementary Data Supplementary Data are available at JB Online.

Funding This research was supported by the National Basic Research Program of China (2011CBA00904), the Key Research Program of the Chinese Academy of Sciences (KSZD-EW-Z-018), and the National Natural Science Foundation of China (30800069, 31070212 and 31370275). Conflict of Interest None declared.

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contributes to the hydrophobic core inside trimers, which might further influence the whole hydrophobicity of LHCII, which in turn, affects the aggregate formation of LHCII. The nearest Chl to aromatic ring of F105 is Chl b607 (the nearest distance is 3.2 A˚). The deprival of the interaction between the porphyrin of Chl b607 and the benzene ring of F105 by mutation (Fig. 5F) might contribute to the differences in the Qy region of the CD spectrum (Fig. 2). The side chain of Q103, however, forms several hydrogen bonds with nearby molecules or residues, including E94, A100, Chl a604 and one water molecule (Fig. 5C). The loss of the hydrogen bond between Q103 and the water molecule by the alanine substitution (Fig. 5D) increases the hydrophobicity of the lumenal side surface, which might induce more Q103A complexes to form aggregate. The dynamic modelling proposed that E94 played great roles in the maintenance of whole helix E partly because of the hydrogen bond between E94 and Q103 (25), which would be the reason for the conformation change indicated by the CD spectra (Fig. 2).

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