Structural basis of assembly of the human T cell receptor-CD3 complex.

The αß T cell receptor (TCR), in association with the CD3γε-CD3δε-CD3ζζ signalling hexamer, is the primary determinant of T cell development and activation, and of immune responses to foreign antigens. The mechanism of assembly of the TCR-CD3 complex remains unknown. Here we report a cryo-electron microscopy structure of human TCRαß in complex with the CD3 hexamer at 3.7 Å resolution. The structure contains the complete extracellular domains and all the transmembrane helices of TCR-CD3. The octameric TCR-CD3 complex is assembled with 1:1:1:1 stoichiometry of TCRαß:CD3γε:CD3δε:CD3ζζ. Assembly of the extracellular domains of TCR-CD3 is mediated by the constant domains and connecting peptides of TCRαß that pack against CD3γε-CD3δε, forming a trimer-like structure proximal to the plasma membrane. The transmembrane segment of the CD3 complex adopts a barrel-like structure formed by interaction of the two transmembrane helices of CD3ζζ with those of CD3γε and CD3δε. Insertion of the transmembrane helices of TCRαß into the barrel-like structure via both hydrophobic and ionic interactions results in transmembrane assembly of the TCR-CD3 complex. Together, our data reveal the structural basis for TCR-CD3 complex assembly, providing clues to TCR triggering and a foundation for rational design of immunotherapies that target the complex.

Mutagenic analysis of the bundle-shaped phycobilisome from Gloeobacter violaceus.

Gloeobacter violaceus is an ancient cyanobacterium as it branches out from the basal position in the phylogenic tree of cyanobacteria. It lacks thylakoid membranes and its unique bundle-shaped type of phycobilisomes (PBS) for light harvesting in photosynthesis are located on the interior side of cytoplasmic membranes. The PBS from G. violaceus have two large linker proteins that are not present in any other PBS, Glr2806, and Glr1262, which are encoded by the genes glr2806 and glr1262, respectively. The location and functions of the linkers Glr2806 and Glr1262 are currently unclear. Here, we report the studies of mutagenetic analysis of glr2806 and the genes of cpeBA, encoding the ß and α subunits of phycoerythrin (PE), respectively. In the mutant lacking glr2806, the length of the PBS rods remains unchanged, but the bundles are less tightly packed as examined by electron microscopy with negative staining. It is also shown that two hexamers are missing in the peripheral area of the PBS core, strongly suggesting that the linker Glr2806 is located in the core area instead of the rods. In the mutant lacking the cpeBA genes, PE is no longer present and the PBS rods have only three layers of phycocyanin hexamers. The construction of deletional mutants in G. violaceus, achieved for the first time, provides critical information for our understanding of its unique PBS and should be useful in studies of other aspects of this interesting organism as well.

Structural and mechanistic insights into the DNA glycosylase AAG-mediated base excision in nucleosome.

The engagement of a DNA glycosylase with a damaged DNA base marks the initiation of base excision repair. Nucleosome-based packaging of eukaryotic genome obstructs DNA accessibility, and how DNA glycosylases locate the substrate site on nucleosomes is currently unclear. Here, we report cryo-electron microscopy structures of nucleosomes bearing a deoxyinosine (DI) in various geometric positions and structures of them in complex with the DNA glycosylase AAG. The apo nucleosome structures show that the presence of a DI alone perturbs nucleosomal DNA globally, leading to a general weakening of the interface between DNA and the histone core and greater flexibility for the exit/entry of the nucleosomal DNA. AAG makes use of this nucleosomal plasticity and imposes further local deformation of the DNA through formation of the stable enzyme-substrate complex. Mechanistically, local distortion augmentation, translation/rotational register shift and partial opening of the nucleosome are employed by AAG to cope with substrate sites in fully exposed, occluded and completely buried positions, respectively. Our findings reveal the molecular basis for the DI-induced modification on the structural dynamics of the nucleosome and elucidate how the DNA glycosylase AAG accesses damaged sites on the nucleosome with different solution accessibility.

Cryo-EM and femtosecond spectroscopic studies provide mechanistic insight into the energy transfer in CpcL-phycobilisomes.

Phycobilisomes (PBS) are the major light harvesting complexes of photosynthesis in the cyanobacteria and red algae. CpcL-PBS is a type of small PBS in cyanobacteria that transfers energy directly to photosystem I without the core structure. Here we report the cryo-EM structure of the CpcL-PBS from the cyanobacterium Synechocystis sp. PCC 6803 at 2.6-Å resolution. The structure shows the CpcD domain of ferredoxin: NADP+ oxidoreductase is located at the distal end of CpcL-PBS, responsible for its attachment to PBS. With the evidence of ultrafast transient absorption and fluorescence spectroscopy, the roles of individual bilins in energy transfer are revealed. The bilin 1Iß822 located near photosystem I has an enhanced planarity and is the red-bilin responsible for the direct energy transfer to photosystem I.

Structural insight into the mechanism of energy transfer in cyanobacterial phycobilisomes.

Phycobilisomes (PBS) are the major light-harvesting machineries for photosynthesis in cyanobacteria and red algae and they have a hierarchical structure of a core and peripheral rods, with both consisting of phycobiliproteins and linker proteins. Here we report the cryo-EM structures of PBS from two cyanobacterial species, Anabaena 7120 and Synechococcus 7002. Both PBS are hemidiscoidal in shape and share a common triangular core structure. While the Anabaena PBS has two additional hexamers in the core linked by the 4th linker domain of ApcE (LCM). The PBS structures predict that, compared with the PBS from red algae, the cyanobacterial PBS could have more direct routes for energy transfer to ApcD. Structure-based systematic mutagenesis analysis of the chromophore environment of ApcD and ApcF subunits reveals that aromatic residues are critical to excitation energy transfer (EET). The structures also suggest that the linker protein could actively participate in the process of EET in both rods and the cores. These results provide insights into the organization of chromophores and the mechanisms of EET within cyanobacterial PBS.

Cryo-EM structures of human GMPPA-GMPPB complex reveal how cells maintain GDP-mannose homeostasis.

GDP-mannose (GDP-Man) is a key metabolite essential for protein glycosylation and glycophosphatidylinositol anchor synthesis, and aberrant cellular GDP-Man levels have been associated with multiple human diseases. How cells maintain homeostasis of GDP-Man is unknown. Here, we report the cryo-EM structures of human GMPPA-GMPPB complex, the protein machinery responsible for GDP-Man synthesis, in complex with GDP-Man or GTP. Unexpectedly, we find that the catalytically inactive subunit GMPPA displays a much higher affinity to GDP-Man than the active subunit GMPPB and, subsequently, inhibits the catalytic activity of GMPPB through a unique C-terminal loop of GMPPA. Importantly, disruption of the interactions between GMPPA and GMPPB or the binding of GDP-Man to GMPPA in zebrafish leads to abnormal brain development and muscle abnormality, analogous to phenotypes observed in individuals carrying GMPPA or GMPPB mutations. We conclude that GMPPA acts as a cellular sensor to maintain mannose homeostasis through allosterically regulating GMPPB.

Structural and functional insights into the tetrameric photosystem I from heterocyst-forming cyanobacteria.

Two large protein-cofactor complexes, photosystem I and photosystem II, are the central components of photosynthesis in the thylakoid membranes. Here, we report the 2.37-Å structure of a tetrameric photosystem I complex from a heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Four photosystem I monomers, organized in a dimer of dimer, form two distinct interfaces that are largely mediated by specifically orientated polar lipids, such as sulfoquinovosyl diacylglycerol. The structure depicts a more closely connected network of chlorophylls across monomer interfaces than those seen in trimeric PSI from thermophilic cyanobacteria, possibly allowing a more efficient energy transfer between monomers. Our physiological data also revealed a functional link of photosystem I oligomerization to cyclic electron flow and thylakoid membrane organization.

Cryo-EM structures of the 80S ribosomes from human parasites Trichomonas vaginalis and Toxoplasma gondii.

As an indispensable molecular machine universal in all living organisms, the ribosome has been selected by evolution to be the natural target of many antibiotics and small-molecule inhibitors. High-resolution structures of pathogen ribosomes are crucial for understanding the general and unique aspects of translation control in disease-causing microbes. With cryo-electron microscopy technique, we have determined structures of the cytosolic ribosomes from two human parasites, Trichomonas vaginalis and Toxoplasma gondii, at resolution of 3.2-3.4 Å. Although the ribosomal proteins from both pathogens are typical members of eukaryotic families, with a co-evolution pattern between certain species-specific insertions/extensions and neighboring ribosomal RNA (rRNA) expansion segments, the sizes of their rRNAs are sharply different. Very interestingly, rRNAs of T. vaginalis are in size comparable to prokaryotic counterparts, with nearly all the eukaryote-specific rRNA expansion segments missing. These structures facilitate the dissection of evolution path for ribosomal proteins and RNAs, and may aid in design of novel translation inhibitors.

Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1.

A key step in ribosome biogenesis is the nuclear export of pre-ribosomal particles. Nmd3, a highly conserved protein in eukaryotes, is a specific adaptor required for the export of pre-60S particles. Here we used cryo-electron microscopy (cryo-EM) to characterize Saccharomyces cerevisiae pre-60S particles purified with epitope-tagged Nmd3. Our structural analysis indicates that these particles belong to a specific late stage of cytoplasmic pre-60S maturation in which ribosomal proteins uL16, uL10, uL11, eL40 and eL41 are deficient, but ribosome assembly factors Nmd3, Lsg1, Tif6 and Reh1 are present. Nmd3 and Lsg1 are located near the peptidyl-transferase center (PTC). In particular, Nmd3 recognizes the PTC in its near-mature conformation. In contrast, Reh1 is anchored to the exit of the polypeptide tunnel, with its C terminus inserted into the tunnel. These findings pinpoint a structural checkpoint role for Nmd3 in PTC assembly, and provide information about functional and mechanistic roles of these assembly factors in the maturation of the 60S ribosomal subunit.