Three structures of PSI-LHCI from Chlamydomonas reinhardtii suggest a resting state re-activated by ferredoxin.

Photosystem I (PSI) from the green alga Chlamydomonas reinhardtii, with various numbers of membrane bound antenna complexes (LHCI), has been described in great detail. In contrast, structural characterization of soluble binding partners is less advanced. Here, we used X-ray crystallography and single particle cryo-EM to investigate three structures of the PSI-LHCI supercomplex from Chlamydomonas reinhardtii. An X-ray structure demonstrates the absence of six chlorophylls from the luminal side of the LHCI belts, suggesting these pigments were either physically absent or less stably associated with the complex, potentially influencing excitation transfer significantly. CryoEM revealed extra densities on luminal and stromal sides of the supercomplex, situated in the vicinity of the electron transfer sites. These densities disappeared after the binding of oxidized ferredoxin to PSI-LHCI. Based on these structures, we propose the existence of a PSI-LHCI resting state with a reduced active chlorophyll content, electron donors docked in waiting positions and regulatory binding partners positioned at the electron acceptor site. The resting state PSI-LHCI supercomplex would be recruited to its active form by the availability of oxidized ferredoxin.

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex.

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl ß-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection.

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35-217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35-338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214-338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26.

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

Cooperative binding of L-trp to human tryptophan 2,3-dioxygenase: resonance Raman spectroscopic analysis.

Tryptophan 2,3-dioxygenase (TDO) is a tetrameric enzyme that catalyses the oxidative cleavage of l-tryptophan (l-Trp) to N-formylkynurenine by the addition of O(2) across the 2,3-bond of the indole ring. This reaction is the first and rate-limiting step in the kynurenine pathway in mammals. In the present study, we measured the conformational changes in the haem pocket of recombinant human TDO (rhTDO) in ferric form that are induced by l-Trp binding using both resonance Raman and optical absorption spectroscopies. The deconvolution analysis of the haem Raman bands at various concentrations of l-Trp revealed that the wild-type enzyme exhibits homotropic cooperativity in l-Trp binding, which was confirmed by a change in the optical absorption spectra. Mutation analysis showed that the Y42F mutant abolished the cooperative binding, and that the H76A mutant considerably reduced the catalytic activity. These data and the inter-subunit contacts reported in the bacterial TDO structure suggest that the Y42 of rhTDO is responsible for the cooperative binding of l-Trp by participating in the active site of the adjacent subunit.