Two lutein molecules in LHCII have different conformations and functions: Insights into the molecular mechanism of thermal dissipation in plants

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1 Biochemical and Biophysical Research Communications 355 (2007) Two lutein molecules in LHCII have different conformations and functions: Insights into the molecular mechanism of thermal dissipation in plants Hanchi Yan a,b,1, Pingfeng Zhang a,b,1, Chao Wang a, Zhenfeng Liu a,2, Wenrui Chang a, * a National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing , China b Graduate University of the CAS, Beijing , China Received 26 January 2007 Available online 7 February 2007 Abstract When LHCII forms aggregates, the internal conformational changes will result in chlorophyll fluorescence quenching. Uncovering the molecular mechanism of this phenomenon will help us to understand how plants dissipate the excess excitation energy through non-photochemical quenching (NPQ) process. The crystal structure of spinach and pea LHCII have been published, and recently, we solved another crystal structure of LHCII from cucumber at 2.66 Å resolution. Here we present the first direct structural evidence indicating that the two lutein(lut) molecules bound in each LHCII monomer have different conformations, Lut621 has a more twisted conformation than that of Lut620. The intimate interaction between the Lut620 and Chla612/Chla611 dimer leads to form a hetero-trimer, which is considered to be a potential quenching site. We also discovered that the dehydration of the LHCII crystals resulted in a notable shrinkage of the crystal unit cell dimensions which was accompanied by a red-shift of the fluorescence emission spectra of the crystals. These phenomena suggest the changes in the crystal packing during dehydration might be the cause of internal conformational changes within LHCII. We proposed a conformational change related NPQ model based on the structure analysis. Ó 2007 Elsevier Inc. All rights reserved. Keywords: LHCII; X-ray structure; Lutein; Conformation; Pigment hetero-trimer; Non-photochemical quenching Plants are able to regulate photosynthesis via the nonphotochemical quenching (NPQ). NPQ is a multi-pathway process that consists of qe (Energy-dependent quenching), qt (phosphorylation-related migration of major LHCII between PSI and PSII known as state transition), and qi (photoinhibitory quenching caused by the slow and reversible inactivation of PSII reaction centers) [1]. It was previously shown that the acidification of the thylokoid lumen [2], the xanthophyll cycle (zeaxanthin * Corresponding author. Fax: address: wrchang@sun5.ibp.ac.cn (W. Chang). 1 These authors contributed equally to this work. 2 Present address: Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA. formation) [3], and the PsbS protein [4] are necessary for qe. One of the focuses of the investigation on the qe mechanism is to reveal the energy quenching sites. The PSII reaction centers [5], the PSII antenna protein complexes [6,7] and PsbS protein [4] have been thought to contain quenching sites, which still remains controversial. In this work, we base on the newly available LHCII structures and discuss the possible molecular mechanism of NPQ observed in LHCII. LHCII is the major peripheral light harvesting pigment protein complex of photosystem II in higher plants, which collects light energy and transfers the excitation energy toward the reaction centers. It is also involved in the distribution of excitation energy between PSI and PSII, and may have a role in photoprotection [6,8]. There are 14 chlorophyll binding sites (8 Chla and 6 Chlb) as well as X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi: /j.bbrc

2 458 H. Yan et al. / Biochemical and Biophysical Research Communications 355 (2007) carotenoid sites (one neoxanthin, two luteins and one xanthophyll-cycle carotenoid) in LHCII [9]. Carotenoids can serve as accessory light harvesting pigments, whereas their functions in the photoprotection and non-radiative dissipation of excess excitation energy are more important for plants [10]. It was thought that zeaxanthin may have a direct role in quenching the excitation energy [3]. Despite the fact that violaxanthin instead of zeaxanthin was observed in the previous two LHCII crystal structures from spinach and pea [9,11], Pascal et al. observed that the chlorophyll fluorescence of the spinach LHCII crystals was actually quenched, implying that there exists in LHCII a NPQ mechanism independent of zeaxanthin and it is related to the conformational changes within LHCII [12]. However, the underlying molecular mechanism is yet unknown. Spectroscopic data indicated that the two lutein molecules bound in each LHCII monomer have different conformations [13]. Nevertheless, the structural differences between the two luteins were not discussed in the previous two reports of LHCII crystal structures [9,11]. Here we have found the first direct evidence from the 2.66 Å resolution cucumber LHCII structure that the two lutein molecules, which are bound symmetrically via a pseudo twofold axis to the two sides of the helix A/B in each LHCII monomer, have different conformations. Additionally, they also have different geometric arrangements and p p interactions with their adjacent chlorophylls (Chla612, Chla603). We suggest that during the conformational changes that are associated with chlorophyll fluorescence quenching, the two p systems in Lut620 and Chla612 can overlap sufficiently to form a pigment hetero-trimer, a potential quencher [10,12]. Furthermore, we will discuss the possible driving force of the conformational changes and the possible mechanism of the excitation energy dissipation. Materials and Methods Comparison of the LHCII crystal structures. The crystal structures of spinach LHCII (pdb code:1rwt) and cucumber LHCII (Yan et al., to be published) were determined in our lab. The coordinate file of the pea LHCII structure model (pdb code:2bhw) was downloaded from the PDB. The structure analysis and comparison was carried out using the graphic software O [14] and LSQMAN [15]. The figures were prepared with Molscript [16] and Raster3D [17]. Preliminary X-ray crystallographic measurements. Three types of cucumber LHCII crystals were tested under 100 K at the beamline 3W1A at BSRF (Beijing, China), with an X-ray wavelength of 0.99 Å and 300 mm distance from the crystal to the Mar345 image plate. The oscillation angle was 0.5, the exposure time for each diffraction image was 10 min. One or two diffraction images were collected at 0, 30, 60, 90, and 120 for each crystal. The data were processed with the program Denzo & Scalepack [18] to obtain crystal unit cell parameters. 77 K fluorescence emission spectrum. Hitachi fluorescence spectrometry F4500 was used to measure 77 K fluorescence emission spectrum. The samples were excited at 435 nm and the spectra from 600 to 800 nm were recorded. We measured the fluorescence emission spectra of LHCII crystals with different growth times and the LHCII preparations that were used for crystallization at different concentrations. The trimeric LHCII sample and crystals were obtained as described before [9]. The experimental spectra were smoothened and fit with two Gaussian components [19]. Results and discussion Two different conformations of lutein molecules in LHCII We initially observed that the two lutein molecules in each monomer have different conformations in the cucumber LHCII crystal structure when we examined the 2.66 Å resolution electronic density map carefully. The conformation of Lut621 (Lut2 in pea LHCII model [11]), which is located around the center of the trimer, is twisted lefthanded along the polyene chain from the lumenal side to the stromal side when compared with that of Lut620 (Lut1 in pea LHCII model [11]) at the peripheral region (Fig. 1a and b). The end ring of Lut621 at the stromal side stretches into the trimerization region at the monomer monomer interface. This end ring and part of its polyene chain are wrapped and restrained in a cavity formed by the chlorin ring and the phytyl tail of Chl602. The ring is also constrained by the indole ring of Trp46 and Chla603 from the adjacent monomer. The hydroxyl group of the ring forms hydrogen bonds with the carboxyl group of the side-chain of Asp47 and the backbone amides of Thr48 and Ala49, thus attaching itself to the N-terminal trimerization region. There are some intra-trimer hydrogen bonds formed in the region, such interactions help to stabilize the peptide conformation and fix the position and orientation of the indole ring of Trp46 (Fig. 1c). The end ring of Lut621 is sterically constrained by the chlorin ring of the Chla603 from the adjacent monomer, which is 3.4 Å away, so it is in close van der Waals contact with the plane of the Trp46 indole ring and could not keep away from it. Therefore, the steric hindrance imposed on Lut621 forces it to twist and orient its ringed plane almost parallel to the indole ring of Trp46 to attain reasonable van der Waals contact between them (3.4 Å). The fixed position and orientation of Trp46 caused by the trimerization of the LHCII monomers is responsible for imposing steric restraints on Lut621. Ruban et al. reported significant changes in lutein-specific regions of the absorption spectrum, circular dichroism and resonance Raman spectrum occurred upon trimerization of LHCII, which all indicated a substantial distortion of one lutein molecule [13]. These spectroscopic data are consistent with the observations that the Lut621 displays a distorted conformation. On the other hand, Lut620 is located at the periphery of the LHCII trimer, hence its conformation remains unaffected upon trimerization. Structure superposition result shows that the overall structure of the LHCII trimers in the three crystal structures are almost the same. There are no obvious deviations in the position and orientation of each individual chlorophyll or carotenoid. The observed conformational difference between Lut621 and Lut620 is present in all three

3 H. Yan et al. / Biochemical and Biophysical Research Communications 355 (2007) Fig. 1. Two lutein molecules have different conformations and different interactions with surrounding chlorophylls. (The figure was prepared with cucumber LHCII coordinate and electronic density map files.) (a) The 2 Fo Fc density map of the 2 lutein molecules at +2.0 r contour; (b) superposition of the two lutein molecules by overlapping the central atoms of polyene (C13, 14, 15, 33, 34, and 35) (left, front view; right, side view); (c) the interaction between the stromal-side head group of Lut621 and the surrounding chlorophylls and peptides. The gray line indicates the monomer monomer interface. All oxygen atoms are represented in red, nitrogen atoms are represented in blue, magnesium atoms in chlorophylls are represented in gold, and the carbon atoms are represented in different colors: yellow in the polypeptide, orange in Lut621, green in Chl602, and light blue in Chla603 and Chlb609 in another monomer; (d) superposition of the Lut621 Chla603 pair onto Lut620 Chla612 pair (upper, side view; lower, top view). (For interpretation of color mentioned in this figure legend the reader is referred to the web version of this article.) structures. Therefore, we propose that such a unique structural feature might be common among the LHCII trimers from all higher plants. The two luteins have different interaction strengths with neighboring chlorophylls Superposition of the two Lut Chla dimers (Lut620 Chla612 and Lut621-Chla603) of cucumber LHCII (achieved by overlaying the tetrapyrroles of Chla603 and Chla612 on top of each other) showed that the relative geometrical arrangement of the p-systems in the Lut Chl dimers is evidently different (Fig. 1d). The conjugated polyene plane of Lut620 is parallel to the chlorin plane of Chla612 and is only about 3.6 Å away. And the C15 atom in the conjugated system of Lut620 is right above the C3B atom of the chlorin p-system of Chla612. This steric arrangement enables their p molecular orbitals overlap sufficiently around C15(Lut620) C3B(Chla612) and a strong p p stacking interaction is generated between the two pigments. The close cofacial arrangement (3.6 Å) of the p-orbitals from the electron donor (carotenoid) and acceptor (chlorophyll) will facilitate the excitation energy transfer from the chlorophyll to the carotenoid via charge separation mechanism [3,20]. In addition, Chla612 forms a very strong excitonic coupling interaction with the nearby Chla611 [9,21], which aids the dispersal of the strong static dipole moment of the [Lut Chl] * dimer (charge-transfer state) produced during the charge transfer [3,20] and stabilizes the subsequent charge-separated state [Lut + Chl ] [3]. The architecture of the Lut620 Chla612/611 hetero-trimer favors charge separation, resulting in the efficient transfer of excitation energy from the chlorophyll to the carotenoid. The Chla611/612 dimer in its capacity as the terminal fluorescence emitter [10,12] is the final exit site of the energy, which makes it convenient for transferring the excess excitation energy to Lut620 from this site. Since the Chla611/ 612 dimer site is at a relatively low energy level [21] and the Lut620 has a higher energy level than Lut621 [13], the transfer of excess energy from Chla612 to Lut620 might occur primarily through the charge separation mechanism rather than the Förster energy transfer mechanism. In the case of the Lut621 Chla603 pair, the conjugated polyene system of Lut621 is not parallel to the chlorin plane of Chla603 and forms a 20 dihedral angel. Additionally, these two conjugated p systems are a little further apart from each other, and the distortion of the conformation of Lut621 has an effect on the distribution of its p electron cloud, leading to weak p p stacking interactions within the Lut Chl dimer. As a result, the efficiency of the energy transfer via the charge transfer mechanism is much lower for the Lut621 Chla603 pair. The different spatial arrangement and interaction between two luteins and its adjacent chlorophylls is also present in the spinach LHCII and pea LHCII structures. These differences might be responsible for the previously observed different roles of the two lutein molecules in accessory light-harvesting [22], 3 Chl * quenching [23] and rapid phase of NPQ [24]. A hypothesized mechanism of lutein-mediated NPQ As mentioned above, the structural feature of the hetero-trimer makes it a strong candidate for one of the quenching sites. Wentworth et al. reported a direct correlation between the chlorophyll fluorescence quenching and a change in the circular dichroism spectroscopic signals of Lut620 and Chla612/611 in LHCII [10], indicating that the quenching is related to the conformational change

4 460 H. Yan et al. / Biochemical and Biophysical Research Communications 355 (2007) within this domain. In order to find out how the conformational change leads to the formation of the quencher, the Lut621 Chla603 dimer of the cucumber LHCII was superimposed on the Lut620 Chla612 pair by overlapping the tetrapyrroles of Chla603 and Chla612 (Fig. 2a). Assuming that the Lut620 Chla612 pair is located at a new position similar to that of the Lut621 Chla603 pair before the conformational change, we can now have a crude idea about how Lut620 moves during the conformational change. As shown by the red arrow in Fig. 2a, Lut620 rotates clockwise by a small angle during the conformational change, forming a strong p p stacking interaction with the Chla612 as a consequence in the present structures. The question then arises if the rotation of Lut620 results in quenching, what forces are responsible for the rotation? We carried out a detailed analysis of the interactions between the LHCII trimers within the icosahedral proteoliposome vesicle of the cucumber LHCII and spinach LHCII crystals. The contact region between the two adjacent trimers located near the lumenal surface consists of two DGDG molecules, the helix D with its bound Chla614, the C-terminal loop (linked to helix D), and the Fig. 2. Proposed conformational change around the putative NPQ site. (a) Stereo view of the proposed quenching site. The phytyl chains of chlorophylls are omitted for clarity. (b) Side view of the trimer trimer interface. (c) Bottom view of the same interface from the lumenal side. Red arrows in (b) and (c) show the effect of the environmental stress on helix D and the C-terminal loop as a result of inter-trimer interactions. (Yellow or red dotted line, hydrogen bond; black dotted line, coordinate bond.) (For interpretation of color mentioned in this figure legend the reader is referred to the web version of this article.)

5 H. Yan et al. / Biochemical and Biophysical Research Communications 355 (2007) CE Loop with its bound Chlb605 from the adjacent trimer (Fig. 2b and c). The carboxylate side chain of Asp215, located at the contact-region terminus of helix D, makes a short 2.6 Å hydrogen bond with the hydroxyl group (O3D) of the galactosyl head of a DGDG molecule; while the side-chain amide oxygen of the Asn218, at the carboxyl-terminal loop, forms a 2.8 Å hydrogen bond with a hydroxyl group (O2E) of the galactosyl group of another DGDG. In addition, extensive close van der Waals contacts (3 Å) and strong repulsive forces within the inter-trimer contact region are observed for Chla614 and Chlb605, DGDG and the chlorophylls, and DGDG with the residues from the helix D or carboxyl terminus. It is likely that the icosahedral vesicle is stressed and contracts under the pressure of crystal packing. The crowding at the trimer trimer interface results in strong hydrogen bonding interactions, and at the same time, gives rise to extensive van der Waals repulsion between close hydrophobic residues from adjacent trimers, which causes helix D and the carboxyl terminal loop to move towards the interior of the trimers (the central cavity on the lumenal-side of the trimer). Furthermore, the two lipid molecules (DGDG) may be capable of aiding the conformational change [25]. On the other hand, the lumenal-side b-ring of the Lut620 forms an intimate interaction with helix D (P205-A214) through a direct strong hydrogen bond (2.7 Å) from its hydroxyl to the carbonyl of Pro205 and another indirect one from the hydroxyl, via the bridge of Gln197 side-chain nitrogen, to the Asn208 side-chain amide oxygen (Fig. 2a and b). Therefore, the movement of helix D has a direct effect on Lut620. In addition, any steric stress imposed on helix D can be easily relayed to Lut620 via the Chla613 and Chla614 which bound to helix D directly or indirectly (Fig. 2a and b). Hence, we propose that this end of Lut620 should move along the direction of helix D when the inter-trimer crowding causes helix D to contract towards the interior. However, due to the steric hindrance from helix B and nearby chlorophyll molecules, especially the strong repulsion force from Met73 of helix B on the central C35 atom of Lut620 (the nearest distance is 3.4 Å), Lut620 can only rotate, causing it to approach the ring of Chla612. Noticeably, Lut620 is located at the periphery of the trimer where there are no strong steric hindrances from rigid groups, it makes the rotation of Lut620 feasible in space; furthermore, the average temperature factors of Lut620 are higher than those of Lut621 in both the cucumber LHCII and spinach LHCII structures (Supplementary Table 1), which indicates a higher mobility for Lut620 and means that the rotation of Lut620 is possible. Will the crystal packing make the icosahedral vesicle stressed and enhance the van der Waals repulsion within the inter-trimer contact region? We used three types of crystals with different growth times to measure the unit cell parameters by doing X-ray diffraction experiments. As shown in Table 1, it is evident that the crystal unit cell dimensions decrease dramatically as the growth time became longer and the crystals were dehydrated. So the answer is yes. Then, how would the chlorophyll fluorescence of the crystals respond to the dehydration? The earlier fluorescence lifetime experiments showed that the fluorescence of the spinach LHCII crystals, which were about 3 4 months old, were actually quenched to a certain level, and the fluorescence emission peak was red-shifted to 700 nm [12]. Interestingly, we recorded the fluorescence emission spectra of the cucumber and spinach LHCII crystals with different growth times, and found that the redshift of the spectra becomes more evident as the growth time increases (Fig. 3). These data indicate a positive correlation between the extent of unit cell shrinkage and the red shift of chlorophyll fluorescence emission of the crystal samples. Since the chlorophyll fluorescence quenching is always accompanied by the red shift of the fluorescence emission peak position from 680 to 700 nm [12], we infer that the more the unit cell shrinked, the bigger portion of the fluorescence peak is shifted to around 700 nm and to a higher extent the fluorescence quenching will occur in the crystal. Such kind of chlorophyll fluorescence quenching is likely to be a tunable process regulated by the reversible conformational change in LHCII. The crystal structures of cucumber and spinach LHCII both represent the structure of LHCII at a quenched state. Although the crystal packing of the pea LHCII is completely different from that of cucumber and spinach LHCII [9,11], we examined the contact region between the adjacent LHCII trimers in the pea LHCII crystal structure carefully, and suggested that the formation of the close interacting hetero-trimer may have been achieved through the crushing force between adjacent LHCII trimers within the crystal. In summary, we propose that: In the fresh crystal of cucumber LHCII and spinach LHCII, the conformation of LHCII is in a relatively relaxed state, and the chlorophyll fluorescence emission spectrum is close to that of free trimeric sample in solution. As the dehydration occurs gradually, the icosahedral proteoliposome vesicle is shrinked, and meanwhile, the interactions between adjacent LHCII trimers on the vesicle become stronger as they were brought closer. The tension drives the adjustment of Table 1 The crystal unit cell parameters of cucumber LHCII crystals with different growth times Growth time 2 Weeks 2 Months a 2 3 Months 1 Year Crystal unit cell parameters a Used for structure determination. a = b = Å, c = Å a = b = Å, c = Å a = b = Å, c = Å a = b = Å, c = Å

6 462 H. Yan et al. / Biochemical and Biophysical Research Communications 355 (2007) Fig. 3. (a) 77 K fluorescence emission spectra of cucumber LHCII trimers in solution state at low concentration ([Chl] = 0.04 mg/ml, black line) and high concentration ([Chl] = 5.5mg/ml, gray line). (b d) 77 K fluorescence emission spectra of cucumber LHCII crystals with different growth times, the black line is the smoothened experimental spectra curve, the gray line is the fit spectra with two Gaussian components at around 680 and 700 nm. (b) About 2 weeks; (c) 2 3 months; and (d) about 1 year. The same results for the spinach LHCII trimers and crystals are not shown here. (For interpretation of color mentioned in this figure legend the reader is referred to the web version of this article.) internal conformations of LHCII trimer, resulting in the rotation of Lut620 toward the Chla612 Chla611 dimer to form a hetero-trimer of pigments. The more straightforward evidence to support our hypothesis will depend on the determination of the LHCII structure at an unquenched state or at a lower quenching level. As for the question that if there exists any force on the thylakoid membrane which will cause a similar conformational change in LHCII as in the crystal and induce the fluorescence quenching in it, we do not have an answer yet, but we believe that more in vivo investigations will be necessary to address it. Acknowledgments This work was financially supported by grants from the National Basic Research Program of China (2006CB806505), the National Natural Science Foundation of China ( ), the Knowledge Innovation Project of CAS and the President Special Foundation of CAS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.bbrc References [1] P. Muller, X.P. Li, K.K. Niyogi, Non-photochemical quenching. A response to excess light energy, Plant Physiol. 125 (2001) [2] B. Demmig-Adams, W.W. Adams Iii, The role of xanthophyll cycle carotenoids in the protection of photosynthesis, Trends Plant Sci. 1 (1996) [3] N.E. Holt, D. Zigmantas, L. Valkunas, X.P. Li, K.K. Niyogi, G.R. Fleming, Carotenoid cation formation and the regulation of photosynthetic light harvesting, Science 307 (2005) [4] X.P. Li, O. Bjorkman, C. Shih, A.R. Grossman, M. Rosenquist, S. Jansson, K.K. Niyogi, A pigment-binding protein essential for regulation of photosynthetic light harvesting, Nature 403 (2000) [5] G. Finazzi, G.N. Johnson, L. Dall Osto, P. Joliot, F.A. Wollman, R. Bassi, A zeaxanthin-independent nonphotochemical quenching mech-

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