Journal of Structural Biology 126, 86–97 (1999) Article ID jsbi.1999.4106, available online at http://www.idealibrary.com on Crystal Structure of a Phycourobilin-Containing Phycoerythrin at 1.90-Å Resolution1 Stephan Ritter,*,2 Roger G. Hiller,† Pamela M. Wrench,† Wolfram Welte,‡ and Kay Diederichs‡,3 *Institut fu¨ r Biophysik und Strahlenbiologie, Universita¨t Freiburg, Albertstrasse 23, D-79104 Freiburg, Germany; †School of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia; and ‡Fakulta¨tfu¨ r Biologie, Universita¨t Konstanz (M656), D-78457 Konstanz, Germany Received December 22, 1998, and in revised form February 16, 1999 INTRODUCTION The structure of R-phycoerythrin (R-PE) from the red alga Griffithsia monilis was solved at 1.90-Å The process of photosynthesis converts light en- resolution by molecular replacement, using the ergy to chemical energy. For the absorption of light, atomic coordinates of cyanobacterial phycocyanin cyanobacteria and red algae use water-soluble light- from Fremyella diplosiphon as a model. The crystal- harvesting complexes, called phycobilisomes, which lographic R factor for the final model is 17.5% (Rfree are attached to the stromal side of the thylakoid 22.7%) for reflections in the range 100–1.90 Å. The membrane. They have a molecular mass of approxi- ␣␤ model consists of an ( )2 dimer with an internal mately 7–15 ϫ 106 Da and transfer the absorbed noncrystallographic dyad and a fragment of the energy with an efficiency of over 95% (Gantt and ␥ ␣ -polypeptide. The -polypeptide (164 amino acid Lipschultz, 1973; Sauer, 1975; Glazer, 1989) to the residues) has two covalently bound phycoerythrobi- photosynthetic reaction center. lins at positions ␣82 and ␣139. The ␤-polypeptide Phycobilisomes are made up of two structural (177 residues) has two phycoerythrobilins bound to residues ␤82 and ␤158 and one phycourobilin cova- subunits: a core complex, located close to the reac- lently attached to rings A and D at residues ␤50 and tion center, and rod-like segments that are attached ␤61, respectively. The electron density of the ␥-poly- to this core. Both subunits contain different phycobili- peptide is mostly averaged out by threefold crystal- proteins in the form of stacked single discs. The lographic symmetry, but a dipeptide (Gly-Tyr) and various phycobiliproteins differ predominantly in one single Tyr could be modeled. These two tyrosine the number and nature of the pigments they carry residues of the ␥-polypeptide are in close proximity and therefore in their absorption spectra. Their to the phycoerythrobilins at position ␤82 of two arrangement is such that those absorbing in the symmetry-related ␤-polypeptides and are related by blue/green region of the spectrum are located at the ␣␤ the same noncrystallographic dyad as the ( )2 end of the rods, while those absorbing in the red dimer. Possible energy transfer pathways are dis- region are located in the core complex. cussed briefly. ௠ 1999 Academic Press The core complex consists predominantly of allo- Key Words: light-harvesting complexes; red algae; 4 phycobiliproteins; protein structure; molecular phycocyanin (APC), with an absorption maximum ␭ replacement. max at approximately 650 nm, while the major constituents of the rods are phycocyanin (PC), with ␭max at 615–620 nm, and phycoerythrin (PE), with ␭max at 495–565 nm (MacColl and Guard-Friar, 1987). The polypeptide composition of cyanobacterial and 1 This work was supported by the Deutsche Forschungsgemein- algal phycobiliproteins is very similar. Two polypep- schaft/SFB 60 and by Macquarie University. tides of about 15 (␣) and 17 kDa (␤) form an (␣␤) 2 Present address: Medizinische Universita¨tsklinik Abt. Sport- heterodimer, which is the basic structural unit in medizin, Hugstetterstrasse 55, D-79106 Freiburg, Germany. 3 To whom correspondence should be addressed. Fax: ϩϩ49 most phycobiliproteins. Each polypeptide of the ba- 7531 883183. E-mail: [email protected]. sic (␣␤) heterodimer includes eight ␣-helices (X, Y, A, 4 Abbreviations used: R-PE, R-phycoerythrin; B-PE, B-phycoery- B, E, F, G, and H). Their nomenclature points to the thrin; C-PC, C-phycocyanin; APC, allophycocyanin; PC, phycocyanin; structural similarity of helices A to H to the globin PE, phycoerythrin; PCB, phycocyanobilin; PXB, phycobiliviolin; PEB, phycoerythrobilin; PUB, phycourobilin; rms, root-mean-square; fold (Schirmer et al., 1985). The positions of these NCS, noncrystallographic symmetry. helices are so conserved within the three-dimen- 1047-8477/99 $30.00 86 Copyright ௠ 1999 by Academic Press All rights of reproduction in any form reserved. STRUCTURE OF R-PHYCOERYTHRIN 87 FIG. 1. Chemical structure of the bilin chromophores (top) PEB and (bottom) doubly linked PUB. The nomenclature of the atoms is indicated. sional structures that most of the more recent phyco- (␣␤)6␥ and a molecular mass of about 240 kDa for biliprotein structure determinations could be done both phycobiliproteins. using the method of molecular replacement. The (␣␤) In R-PE and B-PE all pigments are open-chain heterodimer is arranged around a crystallographic tetrapyrrole chromophores (bilins), which are cova- threefold axis to form an (␣␤)3 trimer. With few lently bound to cysteines (Glazer et al., 1976). There exceptions (Schirmer et al., 1985, 1987) this trimer is are, however, differences in pigmentation. B-PE car- then related by a twofold noncrystallographic symme- ries five phycoerythrobilins (PEBs) per (␣␤), while in try (NCS) to form the (␣␤)6 hexamer. In addition, a R-PE two phycoerythrobilins (see Fig. 1, top) are third polypeptide of varying molecular mass is pre- attached to the ␣-polypeptide, and two phycoerythro- sent and functions as a so-called linker protein (for bilins and one doubly linked phycourobilin (PUB, see reviews see Glazer, 1985, 1989; Huber, 1989; Gross- Fig. 1, bottom) are bound to the ␤-polypeptide. The man et al., 1993). In the case of R-phycoerythrin pigment composition of the ␥-polypeptide varies. For (R-PE) and B-phycoerythrin (B-PE), this linker pro- B-PE four bilins (two PEBs and two PUBs) are tein (␥-polypeptide) is pigmented, has a molecular described (Glazer, 1985; Ficner et al., 1992) while for mass of about 30 kDa, and is tightly bound to the ␣- R-PE the amount of pigments varies from four to five and ␤-polypeptides. This yields a stoichiometry of bilins (PEB and PUB), depending on the organism. 88 RITTER ET AL. Up to three different ␥-polypeptides are observed for Sequence determination, model building, and refinement. The R-PE of some species (Apt et al., 1993; Stadnichuk et sequence was obtained by genetic methods and was deposited with the EMBL database (Accession No. Z98528). An alignment al., 1993). with the sequences of R-PE from Pol. boldii (Roell and Morse, In the past few years the structures of various 1993), B-PE from Por. cruentum (Sidler et al., 1989), and C-PC phycobiliproteins could be solved, showing the confor- from Fremyella diplosiphon (Mazel et al., 1988) is shown in Table mation of the bound chromophores within the pro- II (for review see Apt et al., 1995). The coordinates of the 1CPC tein (for review see Betz, 1997). The conformation of model (Duerring et al., 1991) were taken from the Brookhaven database and used for molecular replacement. Insertions and phycocyanobilin (PCB) was determined by Schirmer deletions relative to 1CPC were modeled with ‘‘O’’ (Jones et al., et al. (1985, 1986, 1987), Duerring et al. (1991), and 1991). For refinement, molecular dynamics and conventional Brejc et al. (1995). The conformation of phycobilivio- energy restrained least-squares refinement procedures on posi- lin (PXB), phycoerythrobilin, and phycourobilin was tional parameters and B factors were used and water molecules determined by Duerring et al. (1990); Ficner et al. were integrated into the model. A bulk solvent correction was included in the refinement procedure. The free R factor (Bru¨ nger, (1992), Ficner and Huber (1993); and Chang et al. 1997) was used to optimize the refinement strategy. (1996); respectively. In this paper we report the solution of the struc- RESULTS ture by molecular replacement and refinement of Structure Solution by Molecular Replacement R-PE from the red alga Griffithsia monilis at 1.90-Å resolution. The model consists of an ␣-polypeptide As the unit cell parameters of R-PE resemble those carrying two PEBs at positions ␣82 and ␣139 and of of 1CPC, a similar packing was expected. This a ␤-polypeptide carrying two PEBs at positions ␤82 assumption was confirmed by a cross-rotation search. and ␤158 and one PUB doubly-linked to residues ␤50 The (␣␤)2 dimer of 1CPC was placed into a triclinic and ␤61. The two polypeptides ␣ and ␤ are arranged unit cell (a ϭ 155 Å, b ϭ 90 Å, c ϭ 100 Å, ␣ϭ90°, ␤ϭ ␥ϭ around a NCS dyad to form an (␣␤)2 dimer as an 90°, 90°) and its Patterson was generated asymmetric unit. In the following, residues on the with reflections from 15 to 4 Å. The 1000 highest NCS-related ␣-polypeptides of the asymmetric unit Patterson vectors were selected within the radius are designated A and K, respectively. Similarly, 45–5 Å. The cross-rotation function was calculated in residues on the ␤-polypeptide are named B and L, 2.5° steps (0 Յ⌰ϩ Ͻ 720°, 0 Յ⌰2 Յ 180°, respectively. 0 Յ⌰Ϫ Յ 120°; see Rao et al., 1980) in Lattman Although the structure of R-PE from G. monilis is (1972) angle space and gave two clear maxima of expected to be closely related to the structure of equal height at Eulerian angles Ϫ40°, 0°, 0° and 90°, B-PE from Porphyridium sordidum and that of R-PE 180°, 0°. These maxima are related by a 180° rota- from Polysiphonia urceolata, it is of interest because tion and correspond to the internal twofold symme- it was refined to high resolution using the native try of the model dimer. The position of this twofold sequence of G.