Structural insights into the mechanism of rhodopsin phosphodiesterase.

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

Assembly mechanism of a supramolecular MS-ring complex to initiate bacterial flagellar biogenesis in Vibrio species.

The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein, and is an essential core structure which is a part of the rotor. The number and location of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly, and contribution of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component FliG facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating the requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed GFP-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was co-expressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations.Importance Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio, by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization and a supramolecular complex assembly.

Structural Dynamics of a Protein Domain Relevant to the Water-Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy.

Photosystem II (PSII) is a multiprotein complex that has a function of light-driven water oxidation. The catalytic site of water oxidation is the Mn4CaO5 cluster, which is bound to the lumenal side of PSII through amino acid residues from the D1 and CP43 proteins and is further surrounded by the extrinsic proteins. In this study, we have for the first time visualized the structural dynamics of the lumenal region of a PSII core complex using high-speed atomic force microscopy (HS-AFM). The HS-AFM images of a PSII membrane fragment showed stepwise dissociation of the PsbP and PsbO extrinsic proteins. Upon subsequent destruction of the Mn4CaO5 cluster, the lumenal domain of CP43 was found to undergo a conformational fluctuation. The observed structural flexibility and conformational fluctuation of the CP43 lumenal domain are suggested to play important roles in the biogenesis of PSII and the photoassembly of the Mn4CaO5 cluster.