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.2015 Apr 29;10(4):e0124580.
doi: 10.1371/journal.pone.0124580. eCollection 2015.

Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM

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Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM

Ravi Raghav Sonani et al. PLoS One..

Abstract

Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to Rwork (Rfree) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Growth pattern (in terms of chlorophyll, vertical bars) and APC production profile (filled circles) ofPhormidium sp. A09DM, grown for 40 days.
Allophycocyanin content was calculated using the Bennet and Bogorad [36] equations.
Fig 2
Fig 2. PAGE analysis of the purified allophycocyanin.
A) Silver stained and zinc acetate stained 15% SDS-PAGE analyses of purifiedPhormidium APC. Protein molecular mass standard are shown in lane M. Only two bands were observed on silver stained and zinc acetate stained SDS-PAGE. These correspond to α- and β- subunits of the purified APC, suggesting also absence of linker peptide in purified APC complex. Nearly 10 μg of APC protein was loaded in each lane.B) Silver stained and zinc acetate stained 12% Native-PAGE of purifiedPhormidium APC further confirm homogeneity of the purified protein.
Fig 3
Fig 3.Phormidium APC exists as trimer of αβ monomers in solution.
A) UV-visible absorption spectrum (cyan) of purifiedPhormidium APC showed major band at 653 nm that suggests formation of trimer in the solution. The fluorescence emission spectrum (green) of purified APC was measured upon excitation at 645 nm.B) The Superdex 200 gel-filtration profile of thePhormidium protein. The APC protein elutes at volume corresponding to an oligomer of 118 kDa. The column was calibrated with gel filtration molecular weight markers (Carbonic anhydrase, 29 kDa; Ovalbumin, 44.3 kDa; Bovine serum albumin, 67 kDa; Apoferritin, 440 kDa; Bovine thyroglobulin, 669 kDa). Elution volumes of the marker proteins are shown in the Figure.
Fig 4
Fig 4. Representative electron density map.
Shown is the fit of a PCB chromophore (ball-and-stick) in 2Fo—Fc, σA-weighted electron-density map drawn at 1.2 σ contour level. The figure was prepared with Chimera suite [45].
Fig 5
Fig 5. Trimer formation of αβ monomers ofPhormidium APC results in close interactions of the chromophores of two subunits.
A) Ribbon model of the trimer. Each αβ monomer is shown in two shades of the same color. The bound chromphores are shown as spheres.B) Chromophores (shown as spheres) are placed at 51Å apart (center-to-center distance) in an αβ monomer. Closer packing of the chromphores (dcenter-to-center ~21Å) from different αβ monomers results due to oligomerization. The figure was prepared using Chimera suite.
Fig 6
Fig 6. Configuration and conformation of the chromophores in the known APC structures.
The atomic coordinates of PCB chromophores in the known APC structures (PDB IDs: 1ALL, 1KN1, 1B33, 2V8A, 2VJT, 3DBJ and 4F0U) were superposed onto the PCB chromophore ofPhormidium APC by Chimera suite.A) Superposed chromophores bound to α-subunitsB) Superposed chromophores bound to β-subunits.
Fig 7
Fig 7. A LigPlus schematic 2D representation of the chromophore-protein interactions.
Residues from α-subunit (shown as XXXnnn(A)) and from β-subunit (shown as XXXnnn(B)) within a distance of 3.9Å from the chromophore atoms are displayed. Non-chromophore residues involved in hydrophobic contact(s) are shown with residue labels. Side chains of residues forming covalent and H-bonds with the chromophore atoms are shown as sticks. H-bonds are shown in green dashed lines. Chromophore bonds are shown in purple.A) Microenvironment around the α-chromophore (labeled Cyc181(A) in the figure).B) Microenvironment around chromophore (Cyc181(B)) covalently bound to Cys-81 of β-subunit. The figure was prepared using LigPlus suite [41].
Fig 8
Fig 8. Multiple sequence alignment of α (A) and β (B) subunits of APC orthologs, and microenvironments of PCB chromophores in the known APC structures.
APCS,Phormidium; 1ALL,Spirulina platensis; 1KN1,Porphyra yezoensis; 2V8A,Thermosynechococcus Elongatus; 2VJT,Gloeobacter Violaceus; 3DBJ,Thermosynechococcus vulcanus; 4F0U,Synechococcus elongatus PCC 7942. Multiple sequence alignment was achieved with Clustal Omega and residues which constitute microenvironments of PCB chromophores were detected using LigPlus suite. Blue shaded residues of α-subunit and cyan shaded residues of β-subunit are within non-bonded distance of 3.9Å from the chromophore atoms covalently linked to α-subunit. The β-subunit residues shaded in red are within non-bonded distance of 3.9Å from any of the chromophore atom covalently linked to β-subunit. Secondary structure of the Phormidium APC, as estimated using STRIDE (http://webclu.bio.wzw.tum.de/stride/; [51]), is also shown (α, α-helices; G, 310 helices; T, Turns). The figure was prepared using Jalview [52]. The γ-N-methylasparagine residue of the β-subunit is marked with a black square.
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