Structure of Flagellar Motor Proteins in Complex Allows for Insights into Motor Structure and Switching

The flagellar motor is one type of propulsion device of motile bacteria. The cytoplasmic ring (C-ring) of the motor interacts with the stator to generate torque in clockwise and counterclockwise directions. The C-ring is composed of three proteins, FliM, FliN, and FliG. Together they form the “switch complex” and regulate switching and torque generation. Here we report the crystal structure of the middle domain of FliM in complex with the middle and C-terminal domains of FliG that shows the interaction surface and orientations of the proteins. In the complex, FliG assumes a compact conformation in which the middle and C-terminal domains (FliG_MC) collapse and stack together similarly to the recently published structure of a mutant of FliG_MC with a clockwise rotational bias. This intramolecular stacking of the domains is distinct from the intermolecular stacking seen in other structures of FliG. We fit the complex structure into the three-dimensional reconstructions of the motor and propose that the cytoplasmic ring is assembled from 34 FliG and FliM molecules in a 1:1 fashion.     

Study of the Protein Complex,Pore Diameter,and Pore-forming Activity of the Borrelia burgdorferi P13 Porin

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.     

Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as Productive Assembly Intermediates in Arabidopsis

Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabidopsis cell cultures with ¹⁵N and isolating mitochondria, we have identified CI subcomplexes through differences in ¹⁵N incorporation into their protein subunits. The 200-kDa subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII₂. In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII₂, indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly.     

Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants

Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = −33 kcal mol⁻¹) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (K_D = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (K_D) and the regulation of cysteine sensitivity of SAT within the CSC.     

Composition and role of tapetal lipid bodies in the biogenesis of the pollen coat of Brassica napus

The composition of the two major lipidic organelles of the tapetum of Brassica napus L. has been determined. Elaioplasts contained numerous small (0.2–0.6 μm) lipid bodies that were largely made up of sterol esters and triacylglycerols, with monogalactosyldiacylglycerol as the major polar lipid. This is the first report in any species of the presence of non-cytosolic, sterol ester-rich, lipid bodies. The elaioplast lipid bodies also contained 34- and 36-kDa proteins which were shown by N-terminal sequencing to be homologous to fibrillin and other plastid lipid-associated proteins. Tapetosomes contained mainly polyunsaturated triacylglycerols and associated phospholipids plus a diverse class of oleosin-like proteins. The pollen coat, which is derived from tapetosomes and elaioplasts, was largely made up of sterol esters and the C-terminal domains of the oleosin-like proteins, but contained virtually no galactolipids, triacylglycerols or plastid lipid-associated proteins. The sterol compositions of the elaioplast and pollen coat were almost identical, consisting of stigmasterol > campestdienol > campesterol > sitosterol ≫ cholesterol, which is consistent with the majority of the pollen coat lipids being derived from elaioplasts. These data demonstrate that there is substantial remodelling of both the lipid and protein components of elaioplasts and tapetosomes following their release into the anther locule from lysed tapetal cells, and that components of both organelles contribute to the formation of the lipidic coating of mature pollen grains. Received: 4 December 1998 / Accepted: 9 February 1999     

The TOR Complex 1 Is Distributed in Endosomes and in Retrograde Vesicles That Form from the Vacuole Membrane and Plays an Important Role in the Vacuole Import and Degradation Pathway

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.     

Lipid,Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels: MITOCHONDRIAL CARRIERS MIGRATE AS MONOMERS NOT DIMERS*

Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein.     

甘蓝型油菜种子中油体的超微结构及蛋白质组分析

以甘蓝型油菜（Brassica napus L.）含油量较高的品种‘ZS11’、含油量中等的品种‘Westar’和‘Topas’以及含油量较低的品种‘ZS10’为实验材料,通过超微结构观察和统计,比较分析不同品种种子中油体形态、大小和数量的差异。研究结果显示,品种‘ZS11’种子子叶细胞油体排列致密,形态较小,大部分油体的直径低于1 μm;而在含油量中等或较低的品种中,种子子叶细胞油体排列均显疏松,其中‘Westar’和‘Topas’的油体较大,而‘ZS10’的油体大小不一。本研究还通过双向电泳分析进一步检测了‘Westar’和‘ZS11’种子中总蛋白和油体蛋白的差异表达情况。结果显示,‘Westar’和‘ZS11’种子总蛋白双向电泳图谱中,表达量具有2倍以上差异的蛋白质点共有57个;其中在‘Westar’中特异表达的种子总蛋白质点有24个,在‘ZS11’中有23个。在上述2个品种油体蛋白双向电泳图谱中,表达量具有2倍以上差异的蛋白质点共有52个,在品种‘Westar’中特异表达的有2个,‘ZS11’中有13个。表明不同含油量的油菜品种种子在油体的结构和蛋白组份上均存在差异。     

Structure of the Type VI Effector-Immunity Complex (Tae4-Tai4) Provides Novel Insights into the Inhibition Mechanism of the Effector by Its Immunity Protein

The type VI secretion system (T6SS), a multisubunit needle-like apparatus, has recently been found to play a role in interspecies interactions. The Gram-negative bacteria harboring T6SS (donor) deliver the effectors into their neighboring cells (recipient) to kill them. Meanwhile, the cognate immunity proteins were employed to protect the donor cells against the toxic effectors. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity pairs. Here, we report the crystal structures of Tae4 from Enterobacter cloacae and Tae4-Tai4 complexes from both E. cloacae and Salmonella typhimurium. Tae4 acts as a dl-endopeptidase and displays a typical N1pC/P60 domain. Unlike Tsi1 (type VI secretion immunity 1), Tai4 is an all-helical protein and forms a dimer in solution. The small angle x-ray scattering study combined with the analytical ultracentrifugation reveal that the Tae4-Tai4 complex is a compact heterotetramer that consists of a Tai4 dimer and two Tae4 molecules in solution. Structure-based mutational analysis of the Tae4-Tai4 interface shows that a helix (α3) of one subunit in dimeric Tai4 plays a major role in binding of Tae4, whereas a protruding loop (L4) in the other subunit is mainly responsible for inhibiting Tae4 activity. The inhibition process requires collaboration between the Tai4 dimer. These results reveal a novel and unique inhibition mechanism in effector-immunity pairs and suggest a new strategy to develop antipathogen drugs.     

Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.     

Building blocks of the nexin-dynein regulatory complex in Chlamydomonas flagella

The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.     

Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra α-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5′-phosphate recognition by the BRCT domains of bacterial NAD⁺-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions.     

Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.     

Structure function analysis of an ADP-ribosyltransferase type III effector and its RNA-binding target in plant immunity

The Pseudomonas syringae type III effector HopU1 is a mono-ADP-ribosyltransferase that is injected into plant cells by the type III protein secretion system. Inside the plant cell it suppresses immunity by modifying RNA-binding proteins including the glycine-rich RNA-binding protein GRP7. The crystal structure of HopU1 at 2.7-Å resolution reveals two unique protruding loops, L1 and L4, not found in other mono-ADP-ribosyltransferases. Site-directed mutagenesis demonstrates that these loops are essential for substrate recognition and enzymatic activity. HopU1 ADP-ribosylates the conserved arginine 49 of GRP7, and this reduces the ability of GRP7 to bind RNA in vitro. In vivo, expression of GRP7 with Arg-49 replaced with lysine does not complement the reduced immune responses of the Arabidopsis thaliana grp7-1 mutant demonstrating the importance of this residue for GRP7 function. These data provide mechanistic details how HopU1 recognizes this novel type of substrate and highlights the role of GRP7 in plant immunity.     

Biochemical and Biophysical Characterization of the Selenium-binding and Reducing Site in Arabidopsis thaliana Homologue to Mammals Selenium-binding Protein 1

The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO₃²⁻) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys²¹ and Cys²² as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO₃²⁻ to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.     

The m Subunit of Murine Translation Initiation Factor eIF3 Maintains the Integrity of the eIF3 Complex and Is Required for Embryonic Development,Homeostasis, and Organ Size Control

Mammalian eIF3 is composed of 13 subunits and is the largest eukaryotic initiation factor. eIF3 plays a key role in protein biosynthesis. However, it is not fully understood how different subunits contribute to the structural integrity and function of the eIF3 complex. Whether eIF3 is essential for embryonic development and homeostasis is also not known. Here, we show that eIF3m null embryos are lethal at the peri-implantation stage. Compound heterozygotes (eIF3m^flox/−) or FABP4-Cre-mediated conditional knock-out mice are lethal at mid-gestation stages. Although the heterozygotes are viable, they show markedly reduced organ size and diminished body weight. Acute ablation of eIF3m in adult mouse liver leads to rapidly decreased body weight and death within 2 weeks; these effects are correlated with a severe decline of protein biogenesis in the liver. Protein analyses reveal that eIF3m deficiency significantly impairs the integrity of the eIF3 complex due to down-regulation of multiple other subunits. Two of the subunits, eIF3f and eIF3h, are stabilized by eIF3m through subcomplex formation. Therefore, eIF3m is required for the structural integrity and translation initiation function of eIF3. Furthermore, not only is eIF3m an essential gene, but its expression level is also important for mouse embryonic development and the control of organ size.     

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg²⁺ into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.