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Crystal Structure of Allophycocyanin from Red AlgaePorphyra yezoensis at 2.2-Å Resolution

1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês

10.1074/jbc.274.24.16945

1083-351X

Jinyu Liu, Tao Jiang, Jiping Zhang, Dongcai Liang,

Biocrusts and Microbial Ecology

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-Å resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 Å, c = 189.4 Å, α = β = 90°, γ = 120°. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 Å. The r.m.s. deviations of bond length and angles are 0.015 Å and 2.9°, respectively.In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers.The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1α84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2β of another monomer. In R-PE and C-PC, chromophore 1α84 only forms 1 hydrogen bond with 2β77 residue in subunit 2β. This result may support and explain great spectrum difference exists between APC trimer and monomer. The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-Å resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 Å, c = 189.4 Å, α = β = 90°, γ = 120°. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 Å. The r.m.s. deviations of bond length and angles are 0.015 Å and 2.9°, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1α84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2β of another monomer. In R-PE and C-PC, chromophore 1α84 only forms 1 hydrogen bond with 2β77 residue in subunit 2β. This result may support and explain great spectrum difference exists between APC trimer and monomer. Phycobilisomes are large supramolecular aggregates attached to the stromal side of the thylakoid membrane in cyanobacteria, red algae, and cryptomonads. These supramolecular aggregates are light-harvesting protein pigment complexes that are composed of phycobiliproteins and linker proteins. Based on the absorption of visible light, the phycobiliproteins can be divided into three main groups: phycoerythrin (PE) 1The abbreviations used are: PE, phycoerythrin; APC, allophycocyanin; PC, phycocyanin; C-PC, C-phycocyanin; PEC, phycoerythrocyanin; APC-PY, allophycocyanin from P. yezoensis ; APC-SP, APC from S. platensis ; C-PC-AQ, C-PC from A. quadruplaticum ; C-PC-FR, C-PC from F. diplosiphon ; R-PE-PU, R-PE from Polysiphonia urceolata ; PCB, phycocyanobilin; α, 1β, 2α, 2β, … 6β stand for the individual subunits of different monomers within the hexamer (according to Schirmer et al. (6Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (211) Google Scholar)); r.m.s., root mean square 1The abbreviations used are: PE, phycoerythrin; APC, allophycocyanin; PC, phycocyanin; C-PC, C-phycocyanin; PEC, phycoerythrocyanin; APC-PY, allophycocyanin from P. yezoensis ; APC-SP, APC from S. platensis ; C-PC-AQ, C-PC from A. quadruplaticum ; C-PC-FR, C-PC from F. diplosiphon ; R-PE-PU, R-PE from Polysiphonia urceolata ; PCB, phycocyanobilin; α, 1β, 2α, 2β, … 6β stand for the individual subunits of different monomers within the hexamer (according to Schirmer et al. (6Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (211) Google Scholar)); r.m.s., root mean square or phycoerythrocyanin (PEC), phycocyanin (PC), and allophycocyanin (APC). With the help of linker proteins, phycobiliproteins form the two distinct structural domains of phycobilisome, the core and the rods. The core, which is composed of three or more core cylinders associated by APC discs, is in proximity of the reaction centers, whereas the rods are attached on the core and are composed of PC discs in the middle and PE or PEC discs on the tip. Light energy is transferred from PE or PEC via PC to APC and finally to the reaction centers (1Glazer A.N. Biochim. Biophys. Acta. 1984; 768: 29-51Crossref Scopus (415) Google Scholar).The crystal structures of several phycobiliproteins have been solved; among them, three are PEs (2Ficner R. Huber R. Eur. J. Biochem. 1992; 218: 103-106Crossref Scopus (82) Google Scholar, 3Ficner R. Lobeck K. Schimdt G. Huber R. J. Mol. Biol. 1992; 228: 935-950Crossref PubMed Scopus (128) Google Scholar, 4Chang W.R. Jiang T. Wan Z.L. Zhang J.P. Yang Z.X. Liang D.C. J. Mol. Biol. 1996; 262: 721-731Crossref PubMed Scopus (113) Google Scholar), three are C-phycocyanins (C-PCs) (5Schirmer T. Bode W. Huber R. Sidler W. Zuber H. J. Mol. Biol. 1985; 184: 257-277Crossref PubMed Scopus (290) Google Scholar, 6Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (211) Google Scholar, 7Duerring M. Schmidt G.B. Huber R. J. Mol. Biol. 1991; 217: 577-592Crossref PubMed Scopus (189) Google Scholar), one is PEC:PEC from Mastigocladus laminosus (8Duerring M. Huber R. Bode W. Ruembeli R. Zuber H. J. Mol. Biol. 1990; 211: 633-644Crossref PubMed Scopus (141) Google Scholar), and one is APC:APC from Spirulina platensis (9Brejc K. Ficner R. Huber R. Steinbacher S. J. Mol. Biol. 1995; 249: 424-440Crossref PubMed Scopus (195) Google Scholar). All these structures are very similar. The basic building block is an αβ monomer composed of α and β subunits (R-phycoerythrin (R-PE) and B-phycoerythrin (B-PE) have a third subunit γ in the center of the molecule); three αβ monomers are arranged around a 3-fold symmetry axis to form an (αβ)3 trimer or two (αβ)3 trimers, which are assembled face to face into an (αβ)6 hexamer.The crystal structure of APC is very special compared with other phycobiliproteins. First, the spectrum difference between APC trimer and its monomer is very large. When APC monomers aggregate to trimer, the absorption spectrum has a 40-nm red shift; the CD spectrum also changes a great deal, and exiton interaction in the trimer of APC was suggested (10Csatorday K. MacColl R. Csizmadia V. Biochemistry. 1984; 23: 6466-6470Crossref PubMed Scopus (55) Google Scholar), whereas the spectrum difference between C-PC monomer and its trimer is not so large as in APC, although phycocyanin has the same α84PCB and β84PCB as APC.Second, the functional unit of APC was thought to be a trimer, whereas the function unit of other phycobiliproteins were hexamer (αβ)6 or (αβ)6γ. Brejc and co-workers solved the structure of APC-SP from blue alga S. platensis(9Brejc K. Ficner R. Huber R. Steinbacher S. J. Mol. Biol. 1995; 249: 424-440Crossref PubMed Scopus (195) Google Scholar) in the unit cell of APC-SP crystal; two trimers are associated in a "back to back" manner that might represent the assembly state of APC in nature. Red alga is higher than blue alga in evolution, so it would be interesting to know the packing of APC from red alga in the unit cell and in nature.Third, in PE and PC, the two trimeric discs are superimposed along a 3-fold axis, but in PC and APC the two discs are connected perpendicularly. The pathway of energy transfer between PC and APC is still unknown.The red algae Porphyra yezoensis is an algae that exists widely in nature. Its phycobilisomes contain R-PE, C-PC, and APC. In this paper we report the crystal structure of APC from P. yezoensis (APC-PY) at 2.2-Å resolution. The organization of APC trimers in the core cylinders of phycobiliproteins and the pathway of energy transfer were discussed.EXPERIMENTAL PROCEDURESCrystallization and data collection of APC-PY was reported earlier (11Liu J.Y. Zhang J.P. Wan Z.L. Liang D.C. Zhang J.P. Wu H.J. Acta Crystallogr. Sec. D. 1998; 54: 662-664Crossref PubMed Scopus (4) Google Scholar). The crystals of APC-PY belong to space group R32 with parameters a = b = 105.3 Å, c = 189.4 Å, α = β = 90°, and γ = 120°.Molecular replacement using program AMoRe (12Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) was carried out using the 2.3-Å structure of APC-SP as a model. Model cell parameters were a = b = c = 150.0 Å, α = β = γ = 90°, integrate radius was 30 Å, and rotation function calculation gave a rather high coefficient solution, α = 60.07, β = 3.06, γ = 88.03, Cc = 20.0. The orientations and positions of one αβ in the asymmetric unit were determined by the translation function with a high correlation coefficient of 66.9%. The R-factor in the range from 10 to 4 Å was 36.1%. After rigid-body refinement, R-factor dropped to 33.1%, and the correlation coefficient increased to 71.2%. The packing of molecules in the unit cell was reasonable.The structure was refined using X-PLOR (13Brünger A. XPLOR, Version 2.1. Yale University, New Haven, CN1990Google Scholar). The consensus sequence was used for the initial model building. Fourier transform and electron density were first calculated in the resolution range of 10 Å to 3.5 Å. Residues that could not be fitted into the electron density map were omitted from the phase calculation in the next refinement cycle. After several cycles of rigid body, positional refinement, and manual model adjustment, the R-factor dropped to 25.4%, and a 2Fo-Fc Fourier map looked quite good. Then the resolution was extended to 2.2 Å. After the chromophores were fitted in the map and followed by several cycles of positional refinement and model adjustment, the electron density improved further. At this stage almost all side chains were well defined except those on the surface. Residue exchanges were carried out at this stage according to the omit map. After several cycles of positional refinement and model adjustment, the R-factor was converged to 24.0%, the individual B-factors were then refined, and the R-factor dropped to 21.5%. 169 water molecules were added to the model according to the Fo-Fc and 2Fo-Fc maps, and the final R-factor of the model was 19.3% (R-free factor was 26.9%) in the range of 10 Å to 2.2 Å. Phycobilisomes are large supramolecular aggregates attached to the stromal side of the thylakoid membrane in cyanobacteria, red algae, and cryptomonads. These supramolecular aggregates are light-harvesting protein pigment complexes that are composed of phycobiliproteins and linker proteins. Based on the absorption of visible light, the phycobiliproteins can be divided into three main groups: phycoerythrin (PE) 1The abbreviations used are: PE, phycoerythrin; APC, allophycocyanin; PC, phycocyanin; C-PC, C-phycocyanin; PEC, phycoerythrocyanin; APC-PY, allophycocyanin from P. yezoensis ; APC-SP, APC from S. platensis ; C-PC-AQ, C-PC from A. quadruplaticum ; C-PC-FR, C-PC from F. diplosiphon ; R-PE-PU, R-PE from Polysiphonia urceolata ; PCB, phycocyanobilin; α, 1β, 2α, 2β, … 6β stand for the individual subunits of different monomers within the hexamer (according to Schirmer et al. (6Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (211) Google Scholar)); r.m.s., root mean square 1The abbreviations used are: PE, phycoerythrin; APC, allophycocyanin; PC, phycocyanin; C-PC, C-phycocyanin; PEC, phycoerythrocyanin; APC-PY, allophycocyanin from P. yezoensis ; APC-SP, APC from S. platensis ; C-PC-AQ, C-PC from A. quadruplaticum ; C-PC-FR, C-PC from F. diplosiphon ; R-PE-PU, R-PE from Polysiphonia urceolata ; PCB, phycocyanobilin; α, 1β, 2α, 2β, … 6β stand for the individual subunits of different monomers within the hexamer (according to Schirmer et al. (6Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (211) Google Scholar)); r.m.s., root mean square or phycoerythrocyanin (PEC), phycocyanin (PC), and allophycocyanin (APC). With the help of linker proteins, phycobiliproteins form the two distinct structural domains of phycobilisome, the core and the rods. The core, which is composed of three or more core cylinders associated by APC discs, is in proximity of the reaction centers, whereas the rods are attached on the core and are composed of PC discs in the middle and PE or PEC discs on the tip. Light energy is transferred from PE or PEC via PC to APC and finally to the reaction centers (1Glazer A.N. Biochim. Biophys. Acta. 1984; 768: 29-51Crossref Scopus (415) Google Scholar). The crystal structures of several phycobiliproteins have been solved; among them, three are PEs (2Ficner R. Huber R. Eur. J. Biochem. 1992; 218: 103-106Crossref Scopus (82) Google Scholar, 3Ficner R. Lobeck K. Schimdt G. Huber R. J. Mol. Biol. 1992; 228: 935-950Crossref PubMed Scopus (128) Google Scholar, 4Chang W.R. Jiang T. Wan Z.L. Zhang J.P. Yang Z.X. Liang D.C. J. Mol. Biol. 1996; 262: 721-731Crossref PubMed Scopus (113) Google Scholar), three are C-phycocyanins (C-PCs) (5Schirmer T. Bode W. Huber R. Sidler W. Zuber H. J. Mol. Biol. 1985; 184: 257-277Crossref PubMed Scopus (290) Google Scholar, 6Schirmer T. Huber R. Schneider M. Bode W. Miller M. Hackert M.L. J. Mol. Biol. 1986; 188: 651-676Crossref PubMed Scopus (211) Google Scholar, 7Duerring M. Schmidt G.B. Huber R. J. Mol. Biol. 1991; 217: 577-592Crossref PubMed Scopus (189) Google Scholar), one is PEC:PEC from Mastigocladus laminosus (8Duerring M. Huber R. Bode W. Ruembeli R. Zuber H. J. Mol. Biol. 1990; 211: 633-644Crossref PubMed Scopus (141) Google Scholar), and one is APC:APC from Spirulina platensis (9Brejc K. Ficner R. Huber R. Steinbacher S. J. Mol. Biol. 1995; 249: 424-440Crossref PubMed Scopus (195) Google Scholar). All these structures are very similar. The basic building block is an αβ monomer composed of α and β subunits (R-phycoerythrin (R-PE) and B-phycoerythrin (B-PE) have a third subunit γ in the center of the molecule); three αβ monomers are arranged around a 3-fold symmetry axis to form an (αβ)3 trimer or two (αβ)3 trimers, which are assembled face to face into an (αβ)6 hexamer. The crystal structure of APC is very special compared with other phycobiliproteins. First, the spectrum difference between APC trimer and its monomer is very large. When APC monomers aggregate to trimer, the absorption spectrum has a 40-nm red shift; the CD spectrum also changes a great deal, and exiton interaction in the trimer of APC was suggested (10Csatorday K. MacColl R. Csizmadia V. Biochemistry. 1984; 23: 6466-6470Crossref PubMed Scopus (55) Google Scholar), whereas the spectrum difference between C-PC monomer and its trimer is not so large as in APC, although phycocyanin has the same α84PCB and β84PCB as APC. Second, the functional unit of APC was thought to be a trimer, whereas the function unit of other phycobiliproteins were hexamer (αβ)6 or (αβ)6γ. Brejc and co-workers solved the structure of APC-SP from blue alga S. platensis(9Brejc K. Ficner R. Huber R. Steinbacher S. J. Mol. Biol. 1995; 249: 424-440Crossref PubMed Scopus (195) Google Scholar) in the unit cell of APC-SP crystal; two trimers are associated in a "back to back" manner that might represent the assembly state of APC in nature. Red alga is higher than blue alga in evolution, so it would be interesting to know the packing of APC from red alga in the unit cell and in nature. Third, in PE and PC, the two trimeric discs are superimposed along a 3-fold axis, but in PC and APC the two discs are connected perpendicularly. The pathway of energy transfer between PC and APC is still unknown. The red algae Porphyra yezoensis is an algae that exists widely in nature. Its phycobilisomes contain R-PE, C-PC, and APC. In this paper we report the crystal structure of APC from P. yezoensis (APC-PY) at 2.2-Å resolution. The organization of APC trimers in the core cylinders of phycobiliproteins and the pathway of energy transfer were discussed. EXPERIMENTAL PROCEDURESCrystallization and data collection of APC-PY was reported earlier (11Liu J.Y. Zhang J.P. Wan Z.L. Liang D.C. Zhang J.P. Wu H.J. Acta Crystallogr. Sec. D. 1998; 54: 662-664Crossref PubMed Scopus (4) Google Scholar). The crystals of APC-PY belong to space group R32 with parameters a = b = 105.3 Å, c = 189.4 Å, α = β = 90°, and γ = 120°.Molecular replacement using program AMoRe (12Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) was carried out using the 2.3-Å structure of APC-SP as a model. Model cell parameters were a = b = c = 150.0 Å, α = β = γ = 90°, integrate radius was 30 Å, and rotation function calculation gave a rather high coefficient solution, α = 60.07, β = 3.06, γ = 88.03, Cc = 20.0. The orientations and positions of one αβ in the asymmetric unit were determined by the translation function with a high correlation coefficient of 66.9%. The R-factor in the range from 10 to 4 Å was 36.1%. After rigid-body refinement, R-factor dropped to 33.1%, and the correlation coefficient increased to 71.2%. The packing of molecules in the unit cell was reasonable.The structure was refined using X-PLOR (13Brünger A. XPLOR, Version 2.1. Yale University, New Haven, CN1990Google Scholar). The consensus sequence was used for the initial model building. Fourier transform and electron density were first calculated in the resolution range of 10 Å to 3.5 Å. Residues that could not be fitted into the electron density map were omitted from the phase calculation in the next refinement cycle. After several cycles of rigid body, positional refinement, and manual model adjustment, the R-factor dropped to 25.4%, and a 2Fo-Fc Fourier map looked quite good. Then the resolution was extended to 2.2 Å. After the chromophores were fitted in the map and followed by several cycles of positional refinement and model adjustment, the electron density improved further. At this stage almost all side chains were well defined except those on the surface. Residue exchanges were carried out at this stage according to the omit map. After several cycles of positional refinement and model adjustment, the R-factor was converged to 24.0%, the individual B-factors were then refined, and the R-factor dropped to 21.5%. 169 water molecules were added to the model according to the Fo-Fc and 2Fo-Fc maps, and the final R-factor of the model was 19.3% (R-free factor was 26.9%) in the range of 10 Å to 2.2 Å. Crystallization and data collection of APC-PY was reported earlier (11Liu J.Y. Zhang J.P. Wan Z.L. Liang D.C. Zhang J.P. Wu H.J. Acta Crystallogr. Sec. D. 1998; 54: 662-664Crossref PubMed Scopus (4) Google Scholar). The crystals of APC-PY belong to space group R32 with parameters a = b = 105.3 Å, c = 189.4 Å, α = β = 90°, and γ = 120°. Molecular replacement using program AMoRe (12Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar) was carried out using the 2.3-Å structure of APC-SP as a model. Model cell parameters were a = b = c = 150.0 Å, α = β = γ = 90°, integrate radius was 30 Å, and rotation function calculation gave a rather high coefficient solution, α = 60.07, β = 3.06, γ = 88.03, Cc = 20.0. The orientations and positions of one αβ in the asymmetric unit were determined by the translation function with a high correlation coefficient of 66.9%. The R-factor in the range from 10 to 4 Å was 36.1%. After rigid-body refinement, R-factor dropped to 33.1%, and the correlation coefficient increased to 71.2%. The packing of molecules in the unit cell was reasonable. The structure was refined using X-PLOR (13Brünger A. XPLOR, Version 2.1. Yale University, New Haven, CN1990Google Scholar). The consensus sequence was used for the initial model building. Fourier transform and electron density were first calculated in the resolution range of 10 Å to 3.5 Å. Residues that could not be fitted into the electron density map were omitted from the phase calculation in the next refinement cycle. After several cycles of rigid body, positional refinement, and manual model adjustment, the R-factor dropped to 25.4%, and a 2Fo-Fc Fourier map looked quite good. Then the resolution was extended to 2.2 Å. After the chromophores were fitted in the map and followed by several cycles of positional refinement and model adjustment, the electron density improved further. At this stage almost all side chains were well defined except those on the surface. Residue exchanges were carried out at this stage according to the omit map. After several cycles of positional refinement and model adjustment, the R-factor was converged to 24.0%, the individual B-factors were then refined, and the R-factor dropped to 21.5%. 169 water molecules were added to the model according to the Fo-Fc and 2Fo-Fc maps, and the final R-factor of the model was 19.3% (R-free factor was 26.9%) in the range of 10 Å to 2.2 Å. We thank Professor Lu-Lu Gui, Institute of Biophysics, Chinese Academy of Sciences and Professor You-Shang Zhang, Institute of Biochemistry, Chinese Academy of Sciences for their support and concern.