Yingang Feng

Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea

Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a β-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

Targeted gene engineering in Clostridium cellulolyticum H10 without methylation

Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative MspI endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C. cellulolyticum mutant H10ΔmspI lost the MspI endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10ΔmspI without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10ΔmspI, double mutants H10ΔmspIΔldh and H10ΔmspIΔack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10ΔmspI, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10ΔmspI constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level.

Metabolic profiles of Nannochloropsis oceanica IMET1 under nitrogen-deficiency stress

To understand the mechanism of lipid accumulation and the corresponding metabolic changes of the microalga Nannochloropsis oceanica IMET1, the lipid content, fatty acid composition and metabolic profile were investigated via batch culture under nutrient deficiency and chemostatic culture under nitrate limitation. The results indicated that the triacylglycerol-neutral lipids were significantly accumulated through an acyl-CoA dependent pathway, while the polar lipids were partially converted to triacylglycerol through an acyl-CoA independent pathway. The fatty acid compositions of the polar lipids changed concurrently with the length of time of the nutrient deficiency, while the fatty acid compositions of the neutral lipids remained nearly consistent. The concentrations of several major osmolytes were significantly changed under chemostatic conditions with different nitrogen concentrations, which reflect the membrane property changes caused by the alteration of the polar lipid composition.

NMR-derived Topology of a GFP-photoprotein Energy Transfer Complex

Förster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca2+-regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously 15N,13C,2H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain 1H-15N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (KD = 0.9 mm). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.

Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented "EF-Hand-Like" Calcium-Binding Phage Lysin

The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.

Native-like Partially Folded Conformations and Folding Process Revealed in the N-terminal Large Fragments of Staphylococcal Nuclease: A Study by NMR Spectroscopy

The N-terminal large fragments of staphylococcal nuclease (SNase), SNase110 (1-110 residues), SNase121 (1-121 residues), and SNase135 (1-135 residues), and the fragment mutants G88W110, G88W121, V66W110 and V66W121 were studied by heteronuclear multidimensional NMR spectroscopy. Ensembles of co-existent native-like partially folded and unfolded states were observed for fragments. The persistent native-like tertiary interaction drives fragments to be in partially folded states, which reveal native-like beta-barrel conformations. G88W and V66W mutations modulate the extent of inherent native-like tertiary interaction in fragment molecules, and in consequence, fragment mutants fold into native-like beta-subdomain conformations. In cooperation with the inherent tertiary interaction, 2 M TMAO (trimethylamine N-oxide) can promote the folding reaction of fragments through the changes of unfolding free energy, and a native-like beta-subdomain conformation is observed when the chain length contains 135 residues. Heterogeneous partially folded conformations of 1-121 and 1-135 fragments due to cis and trans X-prolyl bond of Lys116-Pro117 make a non-unique folding pathway of fragments. The folding reaction of fragments can be characterized as a hierarchical process.

Different impacts of short-chain fatty acids on saturated and polyunsaturated fatty acid biosynthesis in Aurantiochytrium sp. SD116.

Aurantiochytrium is an important docosahexaenoic acid (DHA) producer containing two kinds of fatty acid synthesis pathways, that is, the fatty acid synthase pathway (FAS) for saturated fatty acid synthesis and the polyketide synthase pathway (PKS) for polyunsaturated fatty acid synthesis. To understand the regulation mechanism between the two pathways, the impacts of six short-chain fatty acids on the fatty acid synthesis of Aurantiochytrium sp. SD116 were studied. All short-chain fatty acids showed little effect on the cell growth, but some of them significantly affected lipid accumulation and fatty acid composition. Pentanoic acid and isovaleric acid greatly inhibited the synthesis of saturated fatty acids, whereas the polyunsaturated fatty acid synthesis was not affected. Analysis of malic enzyme activity, which supplied NADPH for saturated fatty acids biosynthesis, indicated that the two fatty acid synthesis pathways can utilize different substrates and possess independent sources of NADPH.

Molecular Basis of Wnt Activation via the DIX Domain Protein Ccd1

The Wnt signaling plays pivotal roles in embryogenesis and cancer, and the three DIX domain-containing proteins, Dvl, Axin, and Ccd1, play distinct roles in the initiation and regulation of canonical Wnt signaling. Overexpressed Dvl has a tendency to form large polymers in a cytoplasmic punctate pattern, whereas the biologically active Dvl in fact forms low molecular weight oligomers. The molecular basis for how the polymeric sizes of Dvl proteins are controlled upon Wnt signaling remains unclear. Here we show that Ccd1 up-regulates canonical Wnt signaling via acting synergistically with Dvl. We determined the crystal structures of wild type Ccd1-DIX and mutant Dvl1-DIX(Y17D), which pack into “head-to-tail” helical filaments. Structural analyses reveal two sites crucial for intra-filament homo- and hetero-interaction and a third site for inter-filament homo-assembly. Systematic mutagenesis studies identified critical residues from all three sites required for Dvl homo-oligomerization, puncta formation, and stimulation of Wnt signaling. Remarkably, Ccd1 forms a hetero-complex with Dvl through the “head” of Dvl-DIX and the “tail” of Ccd1-DIX, depolymerizes Dvl homo-assembly, and thereby controls the size of Dvl polymer. These data together suggest a molecular mechanism for Ccd1-mediated Wnt activation in that Ccd1 converts latent polymeric Dvl to a biologically active oligomer(s).

Transcriptome and gene expression analysis of DHA producer Aurantiochytrium under low temperature conditions

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis, and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future.

Glutathione regulates the transfer of iron-sulfur cluster from monothiol and dithiol glutaredoxins to apo ferredoxin

Holo glutaredoxin (Grx) is a homo-dimer that bridges a [2Fe-2S] cluster with two glutathione (GSH) ligands. In this study, both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur (FeS) cluster to an apo ferredoxin (Fdx) through direct interaction, regardless of FeS cluster stability in holo Grxs. The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH, which inhibits the FeS cluster transfer from holo Grxs to apo Fdx. This phenomenon suggests a novel role of GSH in FeS cluster trafficking.