Heterohexameric Structure Research Articles

Hexameric structure of the flagellar master regulator FlhDC from Cupriavidus necator and its interaction with flagellar promoter DNA

Bacterial flagella are assembled with ∼30 different proteins in a defined order via diverse regulatory systems. In gram-negative bacteria from the Gammaproteobacteria and Betaproteobacteria classes, the transcription of flagellar genes is strictly controlled by the master regulator FlhDC. In Gammaproteobacteria species, the FlhDC complex has been shown to activate flagellar expression by directly interacting with the promoter region in flagellar genes. To obtain the DNA-binding mechanism of FlhDC and determine the conserved and distinct structural features of Betaproteobacteria and Gammaproteobacteria FlhDCs that are necessary for their functions, we determined the crystal structure of Betaproteobacteria Cupriavidus necator FlhDC (cnFlhDC) and biochemically analyzed its DNA-binding capacity. cnFlhDC specifically recognized the promoter DNA of the class II flagellar genes flgB and flhB. cnFlhDC adopts a ring-like heterohexameric structure (cnFlhD4C2) and harbors two Zn-Cys clusters, as observed for Gammaproteobacteria Escherichia coli FlhDC (ecFlhDC). The cnFlhDC structure exhibits positively charged surfaces across two FlhDC subunits as a putative DNA-binding site. Noticeably, the positive patch of cnFlhDC is continuous, in contrast to the separated patches of ecFlhDC. Moreover, the ternary intersection of cnFlhD4C2 behind the Zn-Cys cluster forms a unique protruding neutral structure, which is replaced with a charged cavity in the ecFlhDC structure.

Oligomeric structure of LRRC8/VRAC channels: Arguments for a heptameric conformation.

LRRC8 proteins form large-pore heteromeric volume regulated anion/organic solute channels, VRACs, comprising the LRRC8A subunit and another LRRC8 paralog. Heterologous expression of LRRC8A alone gives rise to homomeric channels with non-physiological properties. Purified LRRC8A protein forms narrow-pore homohexameric channels. Recent cryo-EM structures of purified LRRC8A+LRRC8C proteins revealed a narrow-pore heterohexameric structure with stoichiometries of 5A:1C and 4A:2C. We utilize a chimeric protein approach to define structure-function relationships of LRRC8/VRAC channels. A LRRC8 chimera comprising LRRC8C with 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A, 8C-8A(IL125), gives rise to homomeric channels with normal functional properties. Purified 8C-8A(IL125) protein forms a homoheptameric channel with a large pore diameter similar to that of native LRRC8/VRACs. DCPIB is a LRRC8/VRAC inhibitor. 8C-8A(IL125) homomeric and LRRC8A+LRRC8C heteromeric channels exhibit native DCPIB pharmacology. In contrast, the DCPIB pharmacology of LRRC8A homohexameric channels is grossly abnormal. A chimeric channel comprising LRRC8A and the first extracellular loop (EL1) of LRRC8C, 8A-8C(EL1), has similar abnormal DCPIB pharmacology. Purified 8A-8C(EL1) protein also forms narrow-pore homohexameric channels. An EL1 arginine residue unique to LRRC8A, R103, was proposed to participate in DCPIB inhibition. The R103F mutation greatly reduces DCPIB inhibition of LRRC8A homohexameric channels but has no effect on LRRC8A(R103F)+LRRC8C heteromers. The equivalent EL1 mutation, L105F, also reduces DCPIB inhibition of 8A-8C(EL1) homohexameric channels. In contrast, the L105F mutation has no effect on DCPIB inhibition of 8C-8A(IL125) homoheptameric channels. LRRC8 channels most likely evolved from pannexins, which have a well-established heptameric structure. Based on our functional and structural data and the evolutionary origin of LRRC8 proteins, we propose that native LRRC8/VRAC channels have a heteroheptameric configuration that gives rise to a large pore diameter that accounts for their high organic solute permeability.

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection.

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

Mycobacterium tuberculosis Exploits a Heterohexameric Enoyl-CoA Hydratase Retro-Aldolase Complex for Cholesterol Catabolism.

Cholesterol catabolism plays an important role in Mycobacterium tuberculosis's (Mtb's) survival and persistence in the host. Mtb exploits three β-oxidation cycles to fully degrade the side chain of cholesterol. Five cistronic genes in a single operon encode three enzymes, 3-oxo-4-pregnene-20-carboxyl-CoA dehydrogenase (ChsE1-ChsE2), 3-oxo-4,17-pregnadiene-20-carboxyl-CoA hydratase (ChsH1-ChsH2), and 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA retro-aldolase (Ltp2), to perform the last β-oxidation cycle in this pathway. Among these three enzymes, ChsH1-ChsH2 and Ltp2 form a protein complex that is required for the catalysis of carbon-carbon bond cleavage. In this work, we report the structure of the full length ChsH1-ChsH2-Ltp2 complex based on small-angle X-ray scattering and single-particle electron microscopy data. Mutagenesis experiments confirm the requirement for Ltp2 to catalyze the retro-aldol reaction. The structure illustrates how acyl transfer between enzymes may occur. Each protomer of the ChsH1-ChsH2-Ltp2 complex contains three protein components: a chain of ChsH1, a chain of ChsH2, and a chain of Ltp2. Two protomers dimerize at the interface of Ltp2 to form a heterohexameric structure. This unique heterohexameric structure of the ChsH1-ChsH2-Ltp2 complex provides entry to further understand the mechanism of cholesterol catabolism in Mtb.

Multiple Functions for Mcm2–7 ATPase Motifs during Replication Initiation

The Mcm2-7 replicative helicase is central to all steps of eukaryotic DNA replication. The hexameric ring of Mcm subunits forms six essential ATPases whose contributions to replication initiation remain unclear. Mcm2-7 complexes containing ATPase-motif mutations showed Mcm2-7 ATP binding and hydrolysis are required for helicase loading. Loading-defective Mcm2-7 mutant complexes were defective in initial Mcm2-7 recruitment or Cdt1 release. Comparison with Cdc6 ATPase mutants showed that Cdc6 ATPhydrolysis is not required for helicase loading but instead drives removal of Mcm2-7 complexes that cannot complete loading. A subset of Mcm2-7 ATPase-site mutants completed helicase loading but could not initiate replication. Individual mutants were defective in distinct events during helicase activation, including maintenance of DNA association, recruitment of the GINS helicase activator, and DNAunwinding. Consistent with its heterohexameric structure, our findings show that the six Mcm2-7 ATPase active sites are specialized for different functions during helicase loading and activation.

Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites.

In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.

Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2

Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. NagAa is the ferredoxin-NADP⁺ reductase component containing flavin and plant type [2Fe-2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 °C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12 ± 14.38 U/g and showed an apparent K(m) for salicylate of 102.79 ± 27.20 μM and a similar K(m) value for both NADH and NADPH (59.76 ± 7.81 μM versus 56.41 ± 12.76 μM). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K(m) of 93.54 ± 18.50 μM versus 2,6-dihydroxybenzoate K(m) of 939.80 ± 199.46 μM). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure-function relationships of the ring hydroxylating mono- and di-oxygenase systems.

Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture

DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

Structural Insight into the Dioxygenation of Nitroarene Compounds: the Crystal Structure of Nitrobenzene Dioxygenase

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.