Non-conventional octameric structure of C-phycocyanin.

C-phycocyanin (CPC), a blue pigment protein, is an indispensable component of giant phycobilisomes, which are light-harvesting antenna complexes in cyanobacteria that transfer energy efficiently to photosystems I and II. X-ray crystallographic and electron microscopy (EM) analyses have revealed the structure of CPC to be a closed toroidal hexamer by assembling two trimers. In this study, the structural characterization of non-conventional octameric CPC is reported for the first time. Analyses of the crystal and cryogenic EM structures of the native CPC from filamentous thermophilic cyanobacterium Thermoleptolyngbya sp. O-77 unexpectedly illustrated the coexistence of conventional hexamer and novel octamer. In addition, an unusual dimeric state, observed via analytical ultracentrifugation, was postulated to be a key intermediate structure in the assemble of the previously unobserved octamer. These observations provide new insights into the assembly processes of CPCs and the mechanism of energy transfer in the light-harvesting complexes.

Introduction

Light-harvesting in photosynthesis is a crucial initial step in the conversion of light energy into chemical energy;[1,2] therefore, structural investigation of light-harvesting complexes is important for gaining an understanding of energy transfer mechanisms and developing effective systems of artificial photosynthesis[3-5]. Phycobilisomes (PBSs) are light-harvesting, antenna complexes that are located on the thylakoid membranes of cyanobacteria, red algae, and glaucophytes; they play a key role in the transfer of energy to photosystems I and II[6-9]. The most common hemidiscoidal-type PBSs consist of core and peripheral rod moieties; the core consists of cylindrical substructures of allophycocyanins (APCs), whereas the rod consists of stacked phycocyanins (PCs) and phycoerythrins (PEs)[10]. These phycobiliproteins (PBPs) possess different types of open-chain tetrapyrrole chromophores covalently linked by thioether bonds, resulting in the different absorption properties; the maximum absorptions in APC, PC, and PE are observed at approximately 650–655, 615–620, and 540–570 nm, respectively, which enables PBSs to increase the range of photon absorption from sunlight[7,8]. Recently, cryogenic electron microscopy (cryo-EM) analyses have revealed the complete structures of the PBSs from Griffithsia pacifica and Porphyridium purpureum in high resolution; these analyses illustrate that the interaction between the aromatic amino acids of the linker proteins and the chromophores is likely to be a key factor in the efficient transfer of energy[11,12]. Consequently, the well-organized geometrical arrangement of PBPs and linker proteins in PBSs show substantially high overall quantum efficiency (>90%)[13].

Structurally, PBPs consist of α and β subunits that form the (αβ) monomer; three monomers are then assembled into an (αβ)3 trimer, which is the basic unit of the PBP[8]. C-phycocyanin (CPC), a blue pigment PC mainly observed in cyanobacteria, is a key component in the transfer of energy from the rod to the core[7]. X-ray crystallographic analyses and EM analyses of CPCs have revealed that the quaternary structure of CPC is a closed toroidal (αβ)6 hexamer with D3 symmetry that is thought to be formed by assembling two trimers face-to-face[11,12,14-27]. In solutions, however, the hexameric (αβ)6 state of CPC is only stable in a concentrated phosphate buffer solution at neutral or low pH; it disassembles into trimeric (αβ)3 or even monomeric (αβ) states at low phosphate buffer or low CPC concentrations[28,29]. Therefore, CPC-containing solutions isolated by conventional methods, i.e., column chromatography, usually contain a mixture of monomers, trimers, and hexamers depending on the pH and concentrations of phosphate and CPC[28-30]. Nevertheless, reversible disassembly/reassembly reactions between monomers, trimers, and hexamers have been confirmed in vitro, mainly by analytical ultracentrifugation;[31] thus, it has been suggested that the monomer–trimer–hexamer equilibrium is likely involved in the assembly process of CPCs in vivo[8,23,32]. According to studies on the biosynthetic pathways of CPC, the early events in PBS assembly are also based on the assumption that CPCs have a propensity to form hexameric (αβ)6 states via trimeric (αβ)3 and monomeric (αβ) states[15,28,33].

We recently isolated a filamentous thermophilic cyanobacterium Thermoleptolyngbya sp. O-77 (O-77) from a hot spring in Japan[34-36]. During the course of a study on the photosynthesis of O-77, we isolated a thermostable photosystem II complex from O-77[34]. Thus, we now focus on the isolation of PBPs from O-77 to investigate their light-harvesting properties. In the present study, the native CPC from O-77, namely TlCPC, was isolated and its oligomeric states were determined using X-ray crystallographic and cryo-EM analyses. These structural analyses unexpectedly revealed the coexistence of conventional hexameric (αβ)6 (TlCPC-6) and novel octameric (αβ)8 (TlCPC-8) states, even though TlCPC-6 and TlCPC-8 possessed the same amino acid sequences; notably, PBPs with C4 or D4 symmetries have not previously been reported. In addition, analytical ultracentrifugation of the TlCPC solution showed that the main dissolved species was an unusual dimeric (αβ)2 state. Together with the dissociation free energies of oligomeric (αβ) states (n = 2, 3, 4, 6, and 8), our experimental results indicate that the previously unobserved octamer was presumably assembled from four dimers. To the best of our knowledge, TlCPC-8 is not only the first example of an octameric CPC but also the first reported assemblage of identical subunits into different cyclic oligomers with 3-fold and 4-fold rotation axes.

Results and discussion

Isolation and characterization of TlCPC

Based on genome sequence analysis, O-77 possesses CPC-, APC-, and linker protein-encoding genes but entirely lacks PE-encoding genes, which is similar to several cyanidophytina and cyanobacteria[37]. The genome encodes single genes for CpcA (BAU42407) and CpcB (BAU42408), encoding the α and β subunits for CPC. Since the ApcE gene in O-77 (BAU42084), which encodes the α subunit of APC with a linker in the core of PBS, contains four repeat domains (pfam00427 domains in the Pfam database)[6,38], the PBS complex in O-77 is expected to possess pentacylindrical APC cores[39,40]. It should be noted that a whole structure of this PBS type has, to date, rarely been investigated at high resolution[24,41].

Initially, TlCPC was isolated using a conventional column chromatography technique (see Methods section for details). In SDS-PAGE of TlCPC, approximately 17.5-kDa and 22.5-kDa bands, assignable to the α and β subunits of TlCPC, respectively, were observed (Supplementary Fig. 1). The observed 17.5-kDa band agreed well with the calculated mass of the α subunit (17.4 kDa), whereas the 22.5 kDa band was larger than expected (β subunit: 17.9 kDa). The UV–vis spectrum of TlCPC showed absorption bands at 277, 353, and 615 nm, while the fluorescence spectrum showed an emission band at 644 nm; these are characteristic bands of CPC (Supplementary Fig. 2). The circular dichroism spectrum of TlCPC showed negative Cotton effects with maxima at 220 and 209 nm and a positive Cotton effect with a maximum at 193 nm, which are characteristic bands of α-helix structures (Supplementary Fig. 3). These results were compatible with those previously reported for CPCs and suggested that TlCPC had been successfully isolated[7,14,25].

X-ray crystallographic analysis of TlCPC-6

To elucidate the oligomeric state of TlCPC, the crystallization of TlCPC was examined under various crystallization conditions, which resulted in the formation of many blue single crystals (Supplementary Table 1), where crystal systems were determined in the early data processing stages. The initial screening of X-ray crystallographic analyses revealed that single crystals belonged to the space groups of P2 (No. 2), P21 (No. 4), and P21212 (No. 18), which represented the conventional hexameric (αβ)6 states. TlCPC crystallized in the primitive orthorhombic P21212 was further refined and then the hexameric structure (TlCPC-6) with D3 symmetry was determined at a resolution of 1.65 Å (Fig. 1a, c; Table 1), where the asymmetric unit contained a hexamer. In a comparison with the crystal model of Spirulina platensis CPC (SpCPC; PDB ID: 1GH0), the crystals of which contained two hexamers in the asymmetric unit[17], the root mean square deviation (rmsd) value was 0.68 Å over 1,993 Cα atoms, supporting the formation of a well-established hexameric (αβ)6 state. The α and β subunits of TlCPC-6 shared 68–86% and 65–87% sequence identities, respectively, with the previously reported crystal models of CPCs[14-27]. The X-ray crystallographic analysis of TlCPC-6 showed that phycocyanobilins (PCBs), i.e., the chromophores in CPCs, were covalently bonded at the conserved Cys residues of αCys84 (α84PCB), βCys82 (β82PCB), and βCys153 (β153PCB) (Supplementary Fig. 4). The conformations of PCBs were essentially identical to those in CPCs from Synechococcus elongatus (SeCPC; PDB ID: 1JBO)[19], which were previously determined at high resolution (1.45 Å) with a low Rfree value (Supplementary Fig. 5). The identical conformations of aromatic rings in PCBs also supported the typical absorption and emission spectra observed in TlCPC (Supplementary Fig. 2). Methylation of βAsn72 is a highly conserved post-translational modification in all reported CPCs with few exceptions[20,26] and is thought to play a crucial role in highly efficient energy transfer[42]. In TlCPC-6, the methyl group at the βAsn72 residue could be clearly modeled into an electron density map (Supplementary Fig. 6).

Fig. 1

Crystal models of TlCPC-6 and TlCPC-8.

Top and side views of TlCPC-6 (a, c) and TlCPC-8 (b, d). Dihedral angles of monomers in TlCPC-6 (e) and TlCPC-8 (f). Angles between adjacent monomers in TlCPC-6 (g) and TlCPC-8 (h). The main and side chains of TlCPC-6 and TlCPC-8 are represented by green and cyan ribbon models, respectively. PCBs are represented by grey lines. Yellow lines are guides for measuring angles.

Table 1

Crystallographic data collection and refinement statistics.

(PDB ID)	TlCPC-6 (7EFW)	TlCPC-8 (7EFV)
Data collection
Space group	P22121 (No. 18)	I432 (No. 211)
Cell dimensions
a, b, c (Å)	60.1, 187.4, 210.1	230.0, 230.0, 230.0
α, β, γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0
Resolution (Å)	50.0–1.65 (1.76–1.65)*	50.0–2.77 (2.93–2.77)*
Rmeans	21.2 (106.2)	37.8 (420.0)
CC1/2	99.3 (49.5)	99.8 (55.7)
I/σI	9.5 (1.53)	11.39 (0.91)
Completeness (%)	92.6 (65.1)	100.0 (100.0)
Redundancy	5.8 (2.9)	61.6 (61.7)
Refinement
Resolution (Å)	49.18–1.65	49.03–2.77
No. reflections	264075	26620
Rwork/Rfree (%)	17.0/19.0	19.9/23.8
No. atoms
Protein	15354	5021
Ligand	774	258
Water	2676	—
B-factors
Protein	16.4	68.8
Ligand	16.8	68.9
Water	29.3	—
R.m.s. deviations
Bond lengths (Å)	0.005	0.003
Bond angles (°)	0.985	0.829
Ramachandran plot
Favored (%)	98.3	98.0
Allowed (%)	1.7	2.0
Disallowed (%)	0	0

*Values in parentheses are for a highest-resolution shell.

Determining the crystal packing of CPCs is important for estimating the intra- and inter-CPC energy transfer pathways because orientationally aligned flat-shaped CPCs that are assembled face-to-face in crystals can be regarded as a motif of layered CPCs in the rod moieties of PBSs, even in the absence of linker proteins[15,21,22]. Approximately 90% of the previously reported single crystals from CPCs belong to the space groups of P21 (No. 4), R32 (No. 155), and P63 (No. 173), and most showed orientationally aligned hexameric CPCs in crystals. Only two previous reports indicated that CPCs were crystallized in the orthorhombic space group, with these crystals containing two types of orientation[27,30]. Although TlCPC-6 was also crystallized in the primitive orthorhombic space group (P21212), hexameric CPCs were orientationally aligned in the crystal. In TlCPC-6, the buried surface area between hexamers along the C3 axis, as calculated by the PISA program[43], was 609 Å2 per hexamer, which indicated weak interactions similar to layered CPCs in the rods of PBS (Fig. 2a). Rod-like layered structures of orientationally aligned CPCs, of which C3 axes are shared, have also been observed in CPCs from Microchaete diplosiphon (MdCPC; PDB ID: 1CPC)[14], Thermosynechococcus vulcanus (TvCPC; PDB ID: 1KTP)[18], and Thermosynechococcus elongatus BP-1 (TeCPC; PDB ID: 3L0F). In TlCPC-6, the distances of intraprotein PCB pairs were as follows: α84PCB–β82PCB (20.0 Å), β153PCB–β153PCB (25.8 Å), and α84PCB–α84PCB (26.9 Å) (Supplementary Fig. 7). Additionally, the distances of interprotein PCB pairs were as follows: β82PCB–β82PCB (26.2 Å) and α84PCB–α84PCB (34.4 Å). These values were close to those of the reported CPCs, indicating that the estimated main energy pathways between hexamers were via α84PCB and β82PCB. It is noteworthy that the structure of a double-layered [(αβ)6]2 unit in TlCPC-6 crystal closely resembles that of a double-layered PE–PC unit in PBS from P. purpureum (PDB ID: 6KGX) (Supplementary Fig. 8), supporting that layered CPCs in crystals are structurally correlated with rod moieties of PBSs in vivo.

Fig. 2

Crystal packings of TlCPC-6 and TlCPC-8.

TlCPC-6 (a) and TlCPC-8 (b) in crystals are shown as ribbon representations. Two representatives rod-like [(αβ)6]2 and [(αβ)8]2 structures are shown in red.

Taken together, the aforementioned results show that TlCPC crystallized in the space group P21212 was a conventional hexameric (αβ)6 structure with a layered crystal packing; thus, the distinctive features of TlCPC-6 were hardly observed in comparison with previously reported CPCs.

X-ray crystallographic analysis of TlCPC-8

During X-ray crystallographic analyses, we unexpectedly found crystals that belonged to the extraordinary space group of I centered cubic I432 (No. 211) (Supplementary Table. 1), in which CPCs crystallized in a cubic space group have not previously been reported. X-ray crystallographic analysis revealed that the structure of TlCPC in the space group of I432 at a resolution of 2.77 Å was not a conventional hexamer but rather a novel octamer (TlCPC-8) (Fig. 1b, d; Table 1). This crystal model contained a dimer (αβ)2 in the asymmetric unit and consisted of eight (αβ) monomers to give an extended closed toroidal structure with D4 symmetry. To date, all previously reported PBPs, including APCs, PCs, and PEs, have consisted of basic trimeric (αβ)3 units with C3 symmetry; in contrast, TlCPC-8 possessed a 4-fold rotation axis that could not be directly assembled from trimers or hexamers with 3-fold rotation axes. Notably, TlCPC-8 is the first example of a PBP with a 4-fold rotation axis as well as an octameric CPC.

The inner diameter of ring-shaped TlCPC-8 (45 Å) increased in comparison with that of TlCPC-6 (25 Å) due to the extended toroidal structure of TlCPC-8 (Fig. 1a, b). The structural differences in the (αβ) monomer unit between TlCPC-6 and TlCPC-8 (rmsd value of 2.06 Å over 321 Cα atoms) were larger than those between TlCPC-6 and TvCPC (rmsd value of 0.33 Å over 329 Cα atoms) due to the slight structural conformation changes. In the (αβ) monomer, the helices A, B, E, and F in the α subunit (Aα, Bα, Eα, and Fα), the helices A, B, C, and E in the β subunit (Aβ, Bβ, Cβ, and Eβ), and the loops between these helices (A/Bα, E/Fα, A/Bβ, and B/Cβ) interacted with each other to form a bent structure (Supplementary Fig. 9). The dihedral angle of the (αβ) monomer (αGln70–αArg30–βGly70) in TlCPC-6 was approximately 110°, whereas the equivalent angle in TlCPC-8 was approximately 123° (Fig. 1e, f). In addition, the angle between two adjacent monomers (αGln70–βPro69–βPro69) in TlCPC-6 was approximately 52°, whereas the equivalent angle in TlCPC-8 was approximately 90° (Fig. 1g, h). These results indicate that the structural flexibilities of the subunits and their interfaces presumably affect the assembly process of hexameric (αβ)6 and octameric (αβ)8 states even though both crystal models possess the same amino acid sequence.

In the crystal of TlCPC-8, two octameric (αβ)8 units were stacked along the C4 axis to form a double-layered orientationally aligned structure of [(αβ)8]2, which were further assembled at right angles to each other. Consequently, the overall crystal model of TlCPC-8 possessed a unique framework with exceedingly large void spaces of approximately 100 × 100 × 100 Å3 (Fig. 2b; Supplementary Fig. 10). From an engineering perspective, the topology of this structure resembles zeolite A as well as several types of metal-organic frameworks and polyoxometalates[44,45]; thus, the single crystals of TlCPC-8 could potentially be utilized as catalysts and adsorbents to take advantage of the expected large specific surface area.

Considering [(αβ)8]2 units in the crystal, the layered structures can be regarded as octameric versions of rods (Fig. 2b). The overall conformations of PCBs in TlCPC-8 were essentially identical to those in TlCPC-6 (Supplementary Fig. 5). In TlCPC-8, the distances of intraprotein PCB pairs were as follows: α84PCB–β82PCB (20.7 Å), β153PCB–β153PCB (21.7 Å), and α84PCB–α84PCB (31.6 Å) (Supplementary Fig. 11). These values were similar to those in TlCPC-6, whereas the distance of interprotein PCB pairs of β82PCB–β82PCB (31.7 Å) was increased relative to the equivalent distance in TlCPC-6 (26.2 Å) because a contact area was not observed between octamers by the PISA program, presumably due to the demand for octamers to fit the unique crystal packing of TlCPC-8. Nevertheless, the distances of the interprotein PCB pairs were similar to the shortest distances between chromophores in PE–PE (24.4 Å) and PE–PC (23.8 Å) of PpPBS, indicating the potential intra- and inter-protein energy transfer in TlCPC-8.

As the crystal shapes of TlCPC-6 and TlCPC-8 were different from each other (Supplementary Fig. 12), CPC solutions derived from hexameric (αβ)6 and octameric (αβ)8 states could be prepared by picking up crystals and dissolving them separately. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of these two solutions showed essentially the same two signals assignable to the α and β subunits of TlCPC (Supplementary Fig. 13). In addition, CD spectra of these solutions also showed almost the same ellipticity in a range of 190–250 nm (Supplementary Fig. 14a), and the thermal denaturation midpoints were 71 °C for both solutions (Supplementary Fig. 14b). It should be noted that oligomeric structures were disassembled in these solutions because of the low concentration of CPC[28-32]. These results strongly supported that the subunits composition and physical properties of TlCPC-6 and TlCPC-8 were the same and post-translational modifications did not occur except for the methylation and PCB chromophorylation (Supplementary Figs. 5 and 6). Crystals seemed to be grown one type of morphology in the same drop, and the residual crystallization solution were much clearer than the initial states (Supplementary Fig. 12), indicating the equilibrium between hexameric (αβ)6 and octameric (αβ)8 states during the crystallization process. Collectively, the aforementioned results confirm that the native TlCPC was crystallized in two different morphologies, TlCPC-6 and TlCPC-8, from the same monomeric (αβ) units. Moreover, the coexistence of both structures in concentrated solutions is strongly suggested.

Cryo-EM structure of TlCPC

To avoid the effect of crystallization, the presence of hexameric (αβ)6 and octameric (αβ)8 states was further investigated via cryo-EM analysis. The potassium phosphate buffer solution of TlCPC was applied to a holey carbon grid and then flash-frozen to prepare a cryo-grid for structural determination (Supplementary Note 1). Cryo-EM micrographs and selected reference-free 2D class averages clearly showed two types of ring-shaped oligomeric assemblies in frozen-hydrated specimens as expected (Fig. 3a). Accordingly, each assembly could be subjected to single particle analysis (Fig. 3b, c; Supplementary Figs. 15–18), which resulted in reconstructions of hexameric (αβ)6 and octameric (αβ)8 states with overall resolutions of 3.06 and 3.71 Å, respectively (Fig. 3d–g; Table 2). The EM reconstruction models were essentially isostructural to the crystal models (TlCPC-6: rmsd value of 1.14 Å over 1,923 Cα atoms; TlCPC-8: rmsd value of 1.39 Å over 2,565 Cα atoms) (Supplementary Fig. 19), supporting the coexistence of conventional hexameric (αβ)6 and novel octameric (αβ)8 states in the frozen solution. These results strongly indicated that the concentrated solution of the native TlCPC contained hexamers and octamers. The cryo-EM model of TlCPC-6 was essentially isostructural to the cryo-EM models of PCs in PBSs from G. pacifica (GpPC; PDB ID: 5Y6P) and P. purpureum (PpPC; PDB ID: 6KGX) (GpPC: rmsd value of 1.06 Å over 1,887 Cα atoms; PpPC: rmsd value of 0.96 Å over 1,932 Cα atoms) (Supplementary Fig. 20). Although low-resolution EM structures from CPC and high-resolution cryo-EM structures from PCs in PBSs have been reported, the cryo-EM models of TlCPC-6 and TlCPC-8 are the first examples of high-resolution cryo-EM structures from CPC.

Fig. 3

Cryo-EM analyses of TlCPC-6 and TlCPC-8.

Representative motion-corrected electron micrograph of TlCPCs (a). Typical reference-free 2D class averages from single-particle images of TlCPC-6 (b) and TlCPC-8 (c). Top and side views of cryo-EM density maps of TlCPC-6 at 3.06 Å resolution (d and f) and TlCPC-8 at 3.71 Å resolution (e and g). Contour levels of TlCPC-6 and TlCPC-8 are shown at 0.06 and 0.04, respectively.

Table 2

Cryo-EM data collection, refinement, and validation statistics.

(EMDB/PDB ID)	TlCPC-6 (EMD-31090/7EH8)	TlCPC-8 (EMD-31089/7EH7)
Data collection and processing
Microscope	Talos Arctica	Talos Arctica
Voltage (kV)	200	200
Detector	Falcon 3EC	Falcon 3EC
Magnification	120,000	120,000
Pixel size (Å)	0.88	0.88
Automation software	EPU	EPU
Total exposure (e–/Å2)	50	50
Exposure rate (e–/Å2 fraction)	1.02	1.02
Number of frames	49	49
Defocus range (μm)	−1, −1.5, −2, −2.5	−1, −1.5, −2, −2.5
Number of collected micrograph	2,036	2,036
Number of particles for Class2D	283,980	129,653
Number of particles for Class3D	72,655	23,643
Number of particles for Refine3D	28,120	10,402
Symmetry imposed	D3	D4
Map resolution (Å)	3.06	3.71
FSC threshold	0.143	0.143
Map resolution range (Å)	2.91–3.38	3.47–4.49
Refinement
Refinement programs	PHENIX/Coot	PHENIX/Coot
Initial model used (PDB code)	7EFW	7EFV
Map-to-model resolution (Å)	3.05	3.75
FSC threshold	0.5	0.5
Model resolution range (Å)	2.91–3.38	3.47–4.49
Model composition
Non-hydrogen atoms	15773	20904
Protein residues	2004	2656
Ligands	18	24
B factors (Å2)
Protein	38.8	51.9
Ligand	39.4	51.2
Map-model CC
CC (mask)	0.90	0.86
CC (box)	0.77	0.80
CC (peaks)	0.75	0.75
CC (volume)	0.85	0.83
R.m.s. deviations
Bond lengths (Å)	0.003	0.004
Bond angles (°)	1.051	1.298
Validation
MolProbity score	1.3	1.7
Clash score	5.5	8.7
Poor rotamer (%)	0.79	1.69
Ramachandran plot
Favored (%)	98.2	97.8
Allowed (%)	1.8	2.2
Disallowed (%)	0	0

The local resolution maps of TlCPC-6 and TlCPC-8 showed relatively low resolutions at helices Cα, Dα, Aβ, Bβ, Cβ, Dβ, Fβ, and Gβ and in the loops D/Eα, D/Eβ, F/Gβ, and H/Iβ (Supplementary Fig. 21). These helices and loops also possessed relatively high B-factors in the crystal models of TlCPC-6 and TlCPC-8 (Supplementary Fig. 22). Therefore, the structural fluctuations of these regions are relatively large regardless of the effects of crystal packings (X-ray) and damages induced by freezing processes (cryo-EM).

Geometries of TlCPC-6 and TlCPC-8

Interestingly, the X-ray crystallographic, cryo-EM, and MALDI-TOF mass analyses of TlCPC clearly indicate that different oligomeric states of TlCPC-6 and TlCPC-8 were assembled from the same monomeric (αβ) units as described above. Investigating the structures of homologous proteins with different oligomeric states is crucial to elucidating their functions in metabolism as well as the mechanisms of protein assembly and evolution[46,47]. In general, quaternary structures are conserved among proteins with the same or very high sequence identities because the misassembly of oligomers in vivo is implicated in physiological disorders[46,48]. However, especially in closed circular oligomers with cyclic (C) or dihedral (D) symmetry (n > 2), it has been reported that different oligomeric states from identical subunits, including protective antigens of toxins (n = 7, 8, 38, and 44)[49,50], portal proteins (n = 12 and 13)[51], flagellar motors (n = 32–36)[52], and trp RNA-binding attenuation protein (n = 11 and 12)[53], play important roles in various biological regulations to control diameter and curvature of ring-shaped proteins. These studies have shown that slight conformational changes in secondary and tertiary structures enable the control of rotation angles between adjacent subunits [Δθ, ideally equal to (360/n–360/m)° for oligomers with n- and m-fold rotation axes (n < m)][53]; in contrast, only theoretical studies have been conducted in oligomers with a small number of n-fold rotation axes (n = 3 and 4) because of the requirement of large structural changes (Δθ = 30°) (Supplementary Fig. 23)[54,55]. In this context, TlCPC-6 and TlCPC-8 with their respective D3 and D4 symmetries are the first examples of identical subunits being assembled into different cyclic oligomers with 3- and 4-fold rotation axes.

Analytical ultracentrifugation of TlCPC

To date, size-exclusion chromatography and analytical ultracentrifugation have mainly been utilized to analyze the oligomeric states of CPC in solutions. Thoren et al. reported that a monomeric (αβ) state in solution could be obtained by size-exclusion chromatography, whereas a homogeneous elution of trimeric (αβ)3 or hexameric (αβ)6 states could not be obtained partly due to the aforementioned trimer–hexamer equilibrium of diluted CPC samples in the column[29,32]. On the other hand, analytical ultracentrifugation has long been a powerful tool by which to estimate the oligomeric states of CPCs in solutions. Berns and MacColl previously determined the sedimentation coefficients of CPCs under various conditions and established a widely accepted interpretation as follows: monomeric (αβ), trimeric (αβ)3, and hexameric (αβ)6 species were observed at sedimentation coefficient distributions of 3, 7, and 11 S, respectively[31].

Despite this established consensus, there has been an exceptional observation of dimeric (αβ)2 states: in a report by Neufeld and Riggs, analytical ultracentrifugation of the CPC from S. elongatus PCC 7942 (SeCPC, previously known as Anacystis nidulans) showed monomer–dimer–hexamer equilibrium in solutions[56]. Although X-ray crystallographic analysis later revealed that the SeCPC was a hexameric state in crystals[23], dimeric (αβ)2 states in solutions could be obtained depending on the specific microorganisms or isolation methods.

To investigate the solution states of diluted TlCPC, analytical ultracentrifugation was performed in the present study. Analytical ultracentrifugation of TlCPC (0.92 mg/mL) in potassium phosphate buffer (pH 7.0, 10 mM) gave a major sedimentation coefficient distribution at 5.24 S (s20, = 5.56 S) and minor sedimentation coefficient distributions at 3.15 S (s20, = 3.33 S), 7.97 S (s20, = 8.45 S), and 11.32 S (s20, = 12.00 S) (Supplementary Fig. 24). According to the reported s values, observed species distributed at 3.15, 5.24, and 11.32 S were assignable to monomeric (αβ), dimeric (αβ)2, and hexameric (αβ)6 states, respectively, whereas the species distributed at 7.97 S is observed for the first time here. The molecular weights of these species were calculated using the Svedberg equation as follows: 37.1 kDa (for 3.15 S), 79.8 kDa (for 5.24 S), 149.5 kDa (for 7.97 S), and 253.1 kDa (for 11.32 S). The obtained values of 37.1 and 79.8 kDa were in agreement with the calculated masses of the monomer (37.3 kDa) and dimer (74.7 kDa), respectively, whereas the value of 253.1 kDa was slightly larger than expected (hexamer: 224.1 kDa). Interestingly, the obtained weight of the previously unobserved distribution at 7.97 S (149.5 kDa) was similar to the calculated mass of the tetramer (149.4 kDa). It should be noted that Iso et al. previously reported the presence of tetramers in a solution[57]. Overall, these results indicate that the unusual dimeric (αβ)2 state in the solution was a key intermediate structure in the assembly of the newly observed octameric (αβ)8 state.

Assembly mechanism of TlCPC

Based on the results reported thus far, an assembly mechanism for TlCPC is proposed. The crystal and cryo-EM models of TlCPC-6 and TlCPC-8 clearly showed slight structural differences of monomer–monomer interfaces (helices Gα, Cβ, and Eβ and loop D/Eβ) and α–β subunit interfaces (helices Aα, Bα, Eα, Fα, Aβ, Bβ, Cβ, and Eβ) between TlCPC-6 and TlCPC-8; The superimposition of TlCPC-6 and TlCPC-8 monomers showed that slight structural differences existed at helices Aα, Bα, Aβ, Bβ, Cβ, and Dβ and loops A/Bα, D/Eα, A/Bβ, and D/Eβ (Fig. 1e–h; Supplementary Fig. 25). A possible basis for these structural changes were high B-factors and low local resolutions observed by the X-ray crystallographic and cryo-EM analyses, respectively (Supplementary Figs. 21 and 22), suggesting the relatively flexible protein backbones at these interfaces. Therefore, extended cyclic structures were possible because the flexible interfaces acted as “hinges”, which enabled to control of the curvatures of protein rings although TlCPC-6 and TlCPC-8 possessed the same amino acid sequences. In addition, Adir et al. reported that the hydrogen bond network between αAsp28, βAsn35, and βPCB153 was critical to form hexameric structure in TvCPC[16,23], while TlCPC possesses βSer35 that is rarely observed in the previously reported CPC crystals: (1) the direct hydrogen bond between βAsn35–β153PCB was observed in TvCPC, whereas the indirect hydrogen bond between βSer35–β153PCB via a water molecule was observed in TlCPC, and (2) one of the two indirect hydrogen bonds between βAsn35–αAsp28 in TvCPC was not observed in TlCPC-6, suggesting the destabilization of hexameric structures in TlCPC (Supplementary Fig. 26a). Moreover, the indirect hydrogen bond between αLys32–β153PCB in TvCPC is important to stabilize (αβ) monomer, whereas αGlu32 in TlCPC rather stabilizes a dimeric structure (αβ)2 by interacting with the neighboring β subunit in the dimeric interface (Supplementary Figs. 26b and 27a). These unique residues might be responsible for unusual octamer assembly.

The PISA program calculated the solvation free energies of oligomeric (αβ)2, (αβ)3, (αβ)4, (αβ)6, and (αβ)8 states (ΔGint) as –87.4, –162.3, –204.1, –352.3, and –420.3 kcal/mol, respectively. These values increased as the accessible surface area of each oligomer increased, where the structure of the dimer was assumed to be one-third of the hexamer [contact% = buried surface area / (accessible surface area + buried surface area) = 26%] because a dimer in two-thirds of a trimer is expected to be unstable in solution (contact% = 4%) (Supplementary Fig. 27). The dissociation free energies of oligomeric (αβ)2, (αβ)3, (αβ)4, (αβ)6, and (αβ)8 states into monomeric (αβ) states (ΔG0diss) were also calculated as 4.8, 20.9, 16.0, 54.6, and 20.9 kcal/mol, respectively, clearly indicating that hexamers were thermodynamically more stable than octamers. These results also revealed that trimers were thermodynamically more stable than tetramers, which is consistent with findings that all previously observed CPCs in crystals have been trimers or hexamers. Therefore, the formation of the hexamer from monomers is thought to be the major assembly route for CPCs.

By utilizing the ΔG0diss values, the Gibbs free energies of formation (ΔG0f) between oligomers were calculated to further investigate the assembly process (Fig. 4). Interestingly, the ΔG0f value from tetramers into an octamer was positive (11.1 kcal/mol), which indicated that octamers are difficult to assemble from tetramers, whereas the ΔG0f value from dimers into an octamer was negative. These results supported the proposed monomer–dimer–octamer assembly process as a plausible process by which TlCPC-8 is formed. Therefore, the formation of unusual dimeric (αβ)2 states may be kinetically more favorable than the formation of trimers or tetramers in TlCPC, which supports the conclusion that dimers are key intermediate structures in the assembly of the newly observed octameric (αβ)8 state in TlCPC.

Fig. 4

Proposed oligomerization mechanism in TlCPC.

Monomeric (αβ) structures are represented by bended cuboids. Contact% was calculated by the PISA program based on the monomeric (αβ) units. ΔG0f values were calculated from ΔG0diss values, where the ΔG0f values of –4.8 and –1.4 kcal/mol in the dimer were calculated using the crystal models of TlCPC-6 and TlCPC-8, respectively.

In summary, an unusual octameric (αβ)8 state in TlCPC was presumably formed by (1) adjusting the dihedral angles of flexible monomeric (αβ) units, (2) assembling monomers into kinetically favored dimeric (αβ)2 states, and (3) assembling four dimers into a cyclic structure. Although the key parameter for switching the hexameric and octameric states of TlCPC remains under investigation, one possible factor is that crystallization of the solution, freezing of the solution, and analysis of the solution by ultracentrifugation were performed soon after isolating native CPCs from O-77 at 4–20 °C and kinetically controlled. These in vitro results expand the current perspective of the PBP assembly process in vivo.

Conclusion

In this study, a previously unobserved octameric state of CPC that differed from the conventional hexameric state was discovered for the first time. The existence of non-conventional octameric CPC was confirmed by X-ray crystallographic analysis and the first high-resolution cryo-EM analysis of the native CPC from O-77. Although the monomers in both hexameric and octameric states were essentially identical, the structural analysis indicated that slight conformation changes in potentially flexible interfaces and unique amino acid residues enabled the formation of different oligomeric states from the same primary structures. The unusual dimeric states observed via analytical ultracentrifugation were presumably key intermediate structures in the assembly of the octamer in TlCPC; however, the proposed monomer–dimer–octamer assembly process might be observed in other CPCs because TlCPC shares relatively high sequence identities (α: 68–86%; β: 65–87%) with the previously reported hexameric CPCs in crystals. Since the assembly of identical subunits into symmetrically different oligomeric states is rare and interesting in biology, the experimental results reported in this study are important for understanding not only the assembly process of oligomers but also the evolution process of proteins. Although the biological significance of the octameric structure remains to be clarified, we will focus on it in future studies. We believe that this study provides new insights into the assembly processes of CPCs and PBSs both in vivo and in vitro and sheds new light on the mechanism of energy transfer in the light-harvesting complexes of cyanobacteria.

Methods

Chemicals

Potassium hydroxide, potassium dihydrogen phosphate, sodium chloride, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), and hydrochloric acid were purchased from FUJIFILM Wako Pure Chemical Corporation and used as received.

Purification of CPC

Cells of O-77 were grown according to a previously reported procedure[36]. The cells were harvested and homogenized with 10-mM Tris/HCl buffer at pH 8.0 (buffer A) using NZ-1000 (EYELA, Japan) and then disrupted in an ice bath by sonication three times (2-min sonication at 30 W with a 2-min break) using an Ultrasonic Disruptor UD-200 (TOMY SEIKO, Japan). Cell debris and unbroken cells were removed by centrifugation (5000 g, 20 min, 277 K) using a Himac CR20GII (HITACHI, Japan), and the resulting supernatant was centrifuged at 100,000 g for 1 h using an Optima L-90K (Beckman Coulter, USA). The supernatant of soluble cell extracts was then loaded onto a DEAE Sepharose fast flow column (XK 26/20; GE Healthcare Life Sciences, UK) preequilibrated with buffer A by washing with 500 mL of the same buffer at a flow rate of 10 mL min–1. The TlCPC-containing solution was eluted at 0.15–0.24 M NaCl with 10–19 mS cm–1 ionic conductivity using buffer A alone and buffer A containing 1.0 M NaCl (buffer B) as eluents. Fractions containing TlCPC were combined and diluted threefold with buffer A and the resulting solution was loaded onto a Q Sepharose high-performance column (HR 16/10; GE Healthcare Life Sciences) preequilibrated with buffer A by washing with 150 mL of the same buffer at a flow rate of 4 mL min–1. Blue TlCPC-containing solution was eluted at 0.15–0.20 M NaCl with 13–17 mS cm–1 ionic conductivity using buffers A and B. The fractions containing TlCPC were combined and concentrated using Amicon Ultra-15 50 kDa (Merck, Germany). The resulting concentrated TlCPC was purified using a Superdex 200 prep grade column (HR 16/50; GE Healthcare Life Sciences) and a potassium phosphate buffer solution (pH 7.0, 10 mM) as an eluent. The concentration of the purified TlCPC solution was determined by the following equation[58]:where A615 and A652 are the absorption intensities at 615 and 652 nm, respectively, which were obtained by measuring the UV–vis spectrum.

Spectral analysis

Circular dichroism was measured using Chirascan (Applied Photophysics, UK) with a 0.1-cm quartz cell at 20 °C. The UV–vis spectrum was measured on JASCO V-670 (JASCO, Japan) with a 1-cm quartz cell. The fluorescence spectrum with an excitation wavelength at 436 nm was measured using a FluoroMax-4 Spectrofluorometer (HORIBA Jobin Yvon, NJ).

MALDI-TOF mass spectrometric analysis of CPC solutions were performed using a microflex LT (Bruker Daltonics, Billerica, MA, USA). The saturated sinapinic acid solution in ethanol was deposited onto a ground steel MALDI target plate to form a matrix layer. A mixture of CPC and sinapinic acid in a mixed solvent of acetonitrile and 0.1% trifluoroacetic acid (3/7, v/v) was then dropped on the matrix layer and dried at room temperature (ca. 25 °C). The resulting sample was deposited onto a ground steel MALDI target plate and dried at room temperature (ca. 25 °C).

Analytical ultracentrifugation was performed on an Optima AUC analytical ultracentrifuge (Beckman Coulter) in a four-hole An60Ti rotor at 20 °C. Samples (0.92 mg/mL) in potassium phosphate buffer solution (pH 7.0, 10 mM) were set in aluminum double-sector centerpieces with quartz windows. Concentration profiles were monitored by absorbance at 277 nm because the absorbance at 615 nm is saturated at 0.92 mg/mL of TlCPC. Kao et al. reported that scans of the same sample measured at 620 and 278 nm yielded the same result[59]. The rotor speeds were set at 60,000 rpm. Without intervals between scans, scans were continually recorded until all of the solutes were sedimented at the bottom. Sedimentation velocity data were analyzed by SEDFIT[60] to obtain the distribution of sedimentation coefficients using the continuous c(s) distribution model based on the Lamm equation as follows:where r is the radius from the center of rotation, s is the sedimentation coefficient, t is the time in seconds, and D is the diffusion coefficient. The molecular weight of the corresponding sedimentation coefficient was also calculated by SEDFIT using the Svedberg equation as follows:where M is the molecular weight, is the partial specific volume, ρ is the density of the solution, R is a gas constant, and T is the temperature. The solution density and viscosity of the buffer solution were calculated with SEDNTERP[61] and set at 1.00980 g/mL and 0.01029 P, respectively. The partial specific volume was also calculated with SEDNTERP using the amino acid sequences of the α and β subunits in TlCPC; it was set at 0.73149 mL/g. The frictional ratio was initially set at 1.2 for solving the Lamm equation and then set as a variable for fitting the data. The best-fit frictional ratio was 1.195 with a rmsd of <0.01.

Crystallization, data collection, and structural determination and refinement

The purified TlCPC was crystallized using the sitting-drop vapor diffusion method at 20 °C. Sitting drops contained 200 nL of protein solution mixed with 200 nL of reservoir solution. Two types of crystals (TlCPC-6 and TlCPC-8) were obtained (Supplementary Table 1 and Supplementary Fig. 12). For TlCPC-6 crystals (rhombus or rod), the reservoir condition contained 8% Tacsimate (pH 5.0) and 20% polyethylene glycol 3,350. Prior to data collection, crystals were transferred to a cryoprotectant solution containing polyethylene glycol 3,350 and then flash-cooled to –180 °C. For TlCPC-8 crystals (cube), the reservoir condition contained 1.6-M sodium chloride, 8% polyethylene glycol 6,000, and 20% glycerol, i.e., a cryoprotectant solution. The crystals were then flash-cooled to –180 °C. The cubic crystals of octamers determined by the X-ray crystallographic analysis were also obtained at 42 °C which is the growth temperature of O-77 in this study.

X-ray diffraction data were collected at beamline BL45XU at Spring-8 (Hyogo, Japan) and 100 K with a wavelength of 1.000 Å. These data were processed using the ZOO system[62,63]. Phases were determined by molecular replacement using the program Phaser and search models of monomers of TvCPC (PDB ID: 1I7Y)[16]. Models were built using the program Coot[64], and the program Phenix.refine[65] was used for refinement. The crystal models displayed good geometry when analyzed by MolProbity[66]. In the Ramachandran plots, 98.3% and 1.7% of the residues constituting TlCPC-6 were in the most favored and allowed regions, whereas 98.0% and 2.0% of the residues constituting TlCPC-8 were in the most favored and allowed regions, respectively.

Cryo-EM sample preparation, data collection, and data processing

For cryo-grid preparation, 3 µL of TlCPC samples (6.7 and 10 mg/mL) in potassium phosphate buffer solution (pH 7.0, 10 mM) were applied onto a holey carbon grid (Quantifoil, Cu, R1.2/1.3, 300 mesh). The grid was rendered hydrophilic by a 30 s glow-discharge in the air (11 mA current) with PIB-10 (Vacuum Device Inc., Ibaraki, Japan). The grid was then blotted for 5 sec (blot force 15) at 18 °C and 100% humidity before being flash-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). For automated data collection, 2,036 micrographs were acquired on a Talos Arctica (Thermo Fisher Scientific) microscope operating at 200 kV in nanoprobe mode and using EPU software. The movie micrographs were collected on a 4k × 4k using a Falcon 3EC direct electron detector (electron counting mode) at a nominal magnification of 120,000 (0.88 Å/pixel). Forty-nine movie fractions were recorded at an exposure of 1.02 electrons per Å2 per fraction, which corresponded to a total exposure of 50 e–/Å2. The defocus steps used were –1.0, –1.5, –2.0, and –2.5 µm. The movies were processed by MotionCor2[67], Gctf[68], and RELION3.0[69]. The cryo-EM models were visualized by UCSF chimera[70]. See Supplementary Note 1 and Supplementary Figs. 15–18 for the details of the cryo-EM data processing.