SUPPLEMENTARY INFORMATION Structural insights into energy regulation of light-harvesting complex from spinach CP29 Xiaowei Pan 1, Mei Li 1, Tao Wan 1,2, Longfei Wang 1,2, Chenjun Jia 1,2, Zhiqiang Hou 1,2, Xuelin Zhao 1, Jiping Zhang 1, Wenrui Chang 1* 1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, PR China 2 Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China * Correspondence should be addressed to W.R. Chang (wrchang@sun5.ibp.ac.cn) This PDF file includes: Supplementary Figures 1 to 5 Supplementary Tables 1 to 3 References 1
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Supplementary Figure 1 Electron density maps at 2.8 Å resolution. (a) Region including the Chl pair a615-a611 and their common ligand G3P. Chl a612 in a symmetry related molecule is marked with ' symbol. 2F o F c densities (+1.5σ level) are shown as blue cages. (b to o) Electron densities for each individual Chl molecules are shown with 2F o F c densities (+1.5σ level) in gray and F o F c densities in green (+3σ level) and red (-3σ level). For chlorophyll-binding sites shown in b~i, if it is modeled with Chl b (b~i), strong negative F o F c densities (-3.0σ level) will appear around C7-group, while for those shown in j~m, modeling them as Chl a will lead to strong positive F o F c densities (+3.0σ level) at the oxygen atom position of the C7-groups. In the putative mixed binding site 610, if it is assigned as a Chl a molecule, weak positive F o -F c densities (+2.0σ level) are observed at the end of C7-methyl group (n). However, if it is assigned as a Chl b molecule with full occupancy of C7-formyl oxygen atom, negative F o -F c densities at -2.0σ level appear at the position of this oxygen atom (o). In case that a Chl b at 610 site is assigned with 0.5 occupancy for the C7-formyl oxygen atom, no additional F o -F c densities are observed at that position. Therefore, this site may be occupied by a mixture of Chl a and Chl b. 3
Supplementary Figure 2 Sequence alignment of spinach CP29 and LHCII. The secondary structure of CP29 is shown above the alignment. Red-filled boxes denote identical residues and blue-squared boxes denote homologous residues. The sequence alignment was carried out with the European Bioinformatics Institute (EBI) ClustalW sever 1 and presented using ESPript 2. The PDB code of spinach LHCII is 1RWT 3. 4
Supplementary Figure 3 Polypeptide composition of CP29 crystal and the purified sample used for crystallization. Lane1, CP29 crystal; lane2, purified CP29 sample used for crystallization. The polypeptide of CP29 was degraded non-homogeneously during crystallization, losing about 70 amino acid residues. 5
Supplementary Figure 4 Pigment analysis of CP29 protein by HPLC. Based on the absorption spectrum of each peak fraction (not shown), the pigments in CP29 can be identified as chlorophyll a (Chl a), chlorophyll b (Chl b), neoxanthin (Neo), violaxanthin (Vio) and lutein (Lut). The absorption spectrum of the minor peak between Chl a and Chl b is similar to that of Chl a (not shown). It likely contains an oxidated form of Chl a. 6
Supplementary Figure 5 Room temperature CD spectrum of CP29 protein used for crystallization. 7
Supplementary Table 1 Coordinations of chlorophylls and their interactions with local environment Chls in CP29 Chls in LHCII (PDB code 1RWT) 3 Chls Central ligands Distance between ligands and Mg (Å) H bond to Chl b C7-formyl H bond to Chl C13 1 -keto group Chls in ref 4 Chls Central ligands H bond to Chl b C7-formyl - - - - - - b601 Tyr 24 - a602 Glu 96 OE1 2.15 - - a4 a602 Glu 65 - a603 His 99 NE2 2.26 - Arg 91 NH2 a5 a603 His 68 - a604 Wat 2.19 - Leu 136 N - a604 Wat - - - - - - b605 Val 119 Gln 122 N, Ser 123 N b606 Wat 2.28 Wat (ligand of b607) - b6 (mixed) * b606 Wat Wat (ligand of b607) b607 Wat 2.25 Glu 151 OE1 Lys 126 N - b607 Wat Gln 131 NE2 b608 Wat 2.27 Gln 161 OE1 Arg 101 NE - b608 Wat Leu 148 N a609 Glu 159 OE1 2.16 - His 99 ND1 b5 (mixed) b609 Glu 139 Gln 131 NE2 a/b610 Glu 197 OE1 2.14 - Gly 175 N, Phe 178 O a1 a610 Glu 180 - a611 G3P O2P 2.58 - His 200 ND1 - a611 PG - a612 His 200 NE2 2.13 - - a2 a612 Asn 183 - a613 Gln 214 OE1 2.18 - - a3 (mixed) a613 Gln 197 - b614 His 229 NE2 2.12 Trp 226 NE1 - b3 (mixed) a614 His 212 - a615 G3P O4P 2.19 - Arg 202 NH1 - - - - * mixed in parentheses here and below indicate that the chlorophyll-binding sites are occupied by a mixture of Chl a and Chl b in the predicted model 4. 8
Supplementary Table 2 Interactions between chlorophylls with center-to-center distances less than 10 Å Chl pair a Cosα b Cosβ c R center (Å) d R Mg (Å) e R π (Å) f κ g Qx-Qx κ h Qy-Qy a611-a615-0.94 1.00 7.01 6.65 5.88-0.98 1.00 a604-b606-0.40 0.80 8.08 8.26 4.54-0.97 0.89 a611-a612 0.94-0.85 9.01 9.09 3.72 1.00 1.26 a603-a609 0.86-0.84 9.39 9.42 3.82 0.98 1.29 a613-b614 0.10 0.29 9.22 9.19 3.91-0.69-0.46 b606-b607-0.90 0.86 9.96 9.92 3.15 1.44 0.93 a The Chl pairs with center-to-center distances less than 10 Å. b c d e f g The angle between two Q x transition dipole moments (NA NC). The angle between two Q y transition dipole moments (ND NB). The distance between molecular centers of two Chls. The distance between the central magnesium atoms of two Chls. Closest distance between conjugated π-systems of two Chls. Orientation factor κ between Q x transition dipole moment of two Chls. k ˆ ˆ 3( ˆ ˆ )( ˆ ˆ 1 2 1 r12 2 r )], where 1 and 2 are the normalized transition dipole [ 12 moment vectors and r 12 is the normalized vector between the centers of pigments 1 and 2 5,6. h Orientation factor κ between Q y transition dipole moments of two Chls. 9
Supplementary Table 3 Interactions between carotenoids and chlorophylls Car Chl Rπ (Å) a Atom Car b Atom Chl b Rcenter c Cosα d Cosβ e κ f car-qx κ g car-qy Lut a610 3.61 C30 CHC 9.76 0.37 0.66-1.14-1.05 Lut a612 3.73 C11 CMC 6.57 0.90-0.44 1.03-0.01 Lut a613 3.83 C5 C1B 16.95-0.15-0.69 0.24 1.78 Vio a602 3.58 C34 CBB 10.12 0.32 0.67-1.21-0.95 Vio a603 3.70 C11 CMC 6.38 0.87-0.51 0.98-0.21 Vio a604 4.12 C7 CBB 17.16-0.45-0.54 0.20 1.68 Vio b606 4.24 C7 CBB 18.96-0.27-0.93 0.49 1.79 Vio b607 4.90 C7 CAB 15.66 0.63-0.75 0.14 1.64 Neo a604 4.13 C25 NB 16.47-0.20 0.38 0.00-1.27 Neo b606 4.26 C29 CHB 12.03-0.82-0.17 1.63-0.49 Neo b608 5.10 C15 C2B 8.96 0.78 0.62 1.07-0.09 Interactions between carotenoids and Chls with the closest distance between the two conjugated π-systems within and or about 5 Å are listed in the table. The directions of transition dipole moments of carotenoids and Chls are assigned as in ref 7. a b c d Closest distance between the two conjugated π-systems of carotenoid and Chl. The closest atoms between the two conjugated π-systems of carotenoid and Chl. Distance between molecular centers of carotenoid and Chl. The angle between the transition dipole moment of carotenoid and the Q x transition dipole moment of Chl. e The angle between the transition dipole moment of carotenoid and the Q y transition dipole moment of Chl. f Orientation factor κ between transition dipole moment of carotenoid and Q x transition dipole moment of Chl. g Orientation factor κ between transition dipole moment of carotenoid and Q y transition dipole moment of Chl. 10
Supplementary references 1. Larkin, M.A. et al. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947-2948 (2007). 2. Gouet, P., Courcelle, E., Stuart, D.I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305-308 (1999). 3. Liu, Z. et al. Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428, 287-92 (2004). 4. Bassi, R., Croce, R., Cugini, D. & Sandona, D. Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites. Proc. Natl. Acad. Sci. U.S.A. 96, 10056-61 (1999). 5. Cinque, G., Croce, R., Holzwarth, A. & Bassi, R. Energy transfer among CP29 chlorophylls: calculated Forster rates and experimental transient absorption at room temperature. Biophys. J. 79, 1706-17 (2000). 6. van Amerongen, H. & van Grondelle, R. Understanding the Energy Transfer Function of LHCII, the Major Light-Harvesting Complex of Green Plants. J. Phys. Chem. B 105, 604-617 (2001). 7. Georgakopoulou, S. et al. Understanding the changes in the circular dichroism of light harvesting complex II upon varying its pigment composition and organization. Biochemistry 46, 4745-54 (2007). 11