Light-induced structural adaptation of the bundle-shaped phycobilisome from thylakoid-lacking cyanobacterium Gloeobacter violaceus

Abstract

Gloeobacter diverged from other lineages early in cyanobacterial evolution, preferentially growing under low light intensity conditions. Among cyanobacteria, G. violaceus exhibits unique features, including lack of a thylakoid membrane and bundle-shaped antenna phycobilisomes (PBSs), densely packed and well-organized on the plasma membrane. However, without high-resolution structures, it has remained unclear how G. violaceus PBSs assemble into a bundle-shaped configuration. Here we solve the cryo-EM structures of PBSs from G. violaceus cells cultured under low (Sr-PBS) or moderate (Lr-PBS) light intensity. These structures reveal two unique linker proteins, L_RC^91kDa and L_RC^81kDa, that play a key role in the PBS architecture. Analysis of the bilin arrangement indicates that the bundle-shaped structure allows efficient energy transfer among rods. Moreover, comparison between Lr-PBS and Sr-PBS uncovers a distinct mode of adaption to increased light intensity wherein the ApcA₂-ApcB₃-ApcD layer can be blocked from binding to the core by altering structural elements exclusively found in the G. violaceus L_CM. This study illustrates previously unrecognized mechanisms of assembly and adaptation to varying light intensity in the bundle-shaped PBS of G. violaceus.

Fig. 1. Overall structures of PBS from G. violaceus 7421.

a, b Cryo-EM maps of two types of assemblies of PBS (Lr-PBS and Sr-PBS) from face and bottom views. The rods are colored gray. PBS core is presented in different colors. The solid and dashed black circles are represented whether ApcA₂-ApcB₃-ApcD layer is bond with the core or not in Lr-PBS and Sr-PBS. c Top view of rods. Enlarged view of the boxed area on the right side of (c) shows the cryo-EM map of Rod b after local refinement. The L_R^CpcC1, L_R^CpcC2, and L_RC^91kDa are colored as pink, orange, and lime green. d Enlarged view of the boxed area of (b) shows the cryo-EM map of Rod d after local refinement. The L_RT and L_RC^81kDa are colored as wheat and warm pink.

Fig. 2. Rod-core linker proteins, L_RC^91kDa and L_RC^81kDa.

a Top, diagram of the structural elements of L_RC^91kDa. Bottom, the structure of L_RC^91kDa is represented as a cartoon. Three Pfam00427 domains are presented in different colors. Four helices are highlighted in sky blue. b Interactions between L_RC^91kDa and core. The grooves on the α-subunits that contact the helices are shown in red. c Sequence alignment of the helices of L_RC^91kDa with L_RC from Synechocystis sp. PCC 6803, Synechococcus sp. strain PCC 7002, Nostocaceae PCC 7120, G.pacifica and P.purpureum. The conserved Phenylalanine or Tryptophan is marked as a red triangle. d Top, diagram of the structural elements of L_RC^81kDa. Bottom, the structure of L_RC^91kDa is represented as a cartoon. The Pfam00427-1 domain we solved is presented in megenta. The Pfam00427-2 and -3 are presented in a dashed ellipse. The helix is highlighted in sky blue. e Interactions between L_RC^81kDa and core. The groove on the α-subunit that contacts the helix is shown in red.

Fig. 3. Phylogenetic relationship of L_RC and classifications of Pfam00427.

a The maximum likelihood phylogenetic trees constructed using the full-length protein sequences of L_RC from thylakoid-lacking cyanobacterium G. violaceus 7421, G. kilaueensis JS1, A. panamensis, and crown cyanobacteria Nostocaceae PCC 7120, Synechocystis sp. PCC 6803, Synechococcus sp. strain PCC 7002, A. platensis, and red algae G.pacifica and P.purpureum. Bootstrap values are presented on the tree nodes, and the values higher than 0.6 are shown. b Left, phylogenetic trees constructed using the separated Pfam00427 domains, Pfam00427 domains of L_R and L_RC are labeled in firebrick and black, respectively. The Pfam00427 domains are classified into two types: Type I and II 00427 domains are shown on the purple and light orange background, respectively. The Pfam00427 domains belong to thylakoid-free cyanobacteria are marked with a red star. Right, the structures of type I and II 00427 domains are shown as a cartoon. c Structural alignment of the type I and II 00427 domains. Region of structural change is shown with 70% transparency. Enlarged view of the boxed area of c shows the altered structures including key aromatic residues, representative helix of type II 00427 domain, as well as the shift of bilins.

Fig. 4. Interactions of the linker proteins L_R^CpcC2 and L_RC^91kDa with chromophores in the rod Rb.

a Overall structure of the rod Rb with the hexamers shown in surface representation and the linker proteins shown in cartoon representation. b Structure of the layer Rb2I. Proteins and bilins are shown in cartoon and sticks representations, respectively. Three β subunits are colored differently, and the β82 PCBs are boxed and analyzed in (c–e). c No interactions between the L_R^CpcC2 and the bilin ^Rb2Iβ₁⁸². d The interactions between F224, F235 and F239 and the bilin ^Rb2Iβ₂⁸². e The interactions between Y118 and ^Rb2Iβ₃⁸². f Structure of the layer Rb1I. Proteins and bilins are shown in cartoon and sticks representations, respectively. Three β subunits are colored differently, and the β82 PCBs are boxed and analyzed in (g–i). g The interactions between F510, F512, F521 and F528 and the bilin ^Rb1Iβ₁⁸². h The interactions between Y404 and the bilin ^Rb1Iβ₂⁸². i The interactions between F474 and the bilin ^Rb1Iβ₃⁸².

Fig. 5. Structural differences between L_CM/Lr-PBS and L_CM/Sr-PBS.

a Structural alignment of L_CM/Lr-PBS and L_CM/Sr-PBS. L_CM/Lr-PBS and L_CM/Sr-PBS are presented in hot pink and green colors. ApcA2-ApcB3-ApcD layer attached with L_CM/Lr-PBS is shown as a surface view. The altered structures (loop1, loop2, and bulge) are highlighted in dashed rectangles. b Enlarged view of the boxed area of (a) shows that the structural change of loop1 reduces the interactions between loop1 and ApcA. c Electrostatic interaction between loop1/Lr-PBS and ApcA. d Enlarged view of the structural change of loop2 shows the absence of the interactions between loop2/Sr-PBS and L_C. loop2/Sr-PBS could form a steric hindrance to prevent L_C binding with the core. e Three successive residues (R238-S239-D240) of α^LCM could form a “bulge” that would occupy the position of the ApcD.

Fig. 6. Possible excitation energy transfer (EET) pathways.

a Estimated maps of the energy transfer pathways among bilins based on orientation factor (κ²) and distances (d). The colors correspond to higher (red), middle (blue), and lower (gray) κ²/d⁶ values, which are used to represent the EET rates. The key bilins of rods are shown as spheres in orange color. Bilins that are more than 44 Å are omitted. The gray and black arrows are represented as the rod-core and inner-core bottleneck, respectively. The energy transfer from top B/B’ to bottom A/A’ is high, represented as red lightning. The absent bilins and pathways in Sr-PBS are shown as yellow spheres and dashes, respectively. The terminal emitters ^A3α_LCM¹⁹⁶ (^A3α_LCM’¹⁹⁶) and ^A4α_ApcD⁸¹ (^A4α_ApcD’⁸¹) are shown in sphere representation. b Top view of the energy transfer pathways. The rods are represented as circles in different colors. EET exchange among the rods is represented by black dashed arrows. c The absence of ApcA2-ApcB3-ApcD layer and EET pathways in Sr-PBS is shown as a surface representation. The corresponding bilins are shown as sticks.