Tingting Ran

Cross-protomer interaction with the photoactive site in oligomeric proteorhodopsin complexes.

Proteorhodopsins (PRs), members of the microbial rhodopsin superfamily of seven-transmembrane-helix proteins that use retinal chromophores, comprise the largest subfamily of rhodopsins, yet very little structural information is available. PRs are ubiquitous throughout the biosphere and their genes have been sequenced in numerous species of bacteria. They have been shown to exhibit ion-pumping activity like their archaeal homolog bacteriorhodopsin (BR). Here, the first crystal structure of a proteorhodopsin, that of a blue-light-absorbing proteorhodopsin (BPR) isolated from the Mediterranean Sea at a depth of 12 m (Med12BPR), is reported. Six molecules of Med12BPR form a doughnut-shaped C6 hexameric ring, unlike BR, which forms a trimer. Furthermore, the structures of two mutants of a related BPR isolated from the Pacific Ocean near Hawaii at a depth of 75 m (HOT75BPR), which show a C5 pentameric arrangement, are reported. In all three structures the retinal polyene chain is shifted towards helix C when compared with other microbial rhodopsins, and the putative proton-release group in BPR differs significantly from those of BR and xanthorhodopsin (XR). The most striking feature of proteorhodopsin is the position of the conserved active-site histidine (His75, also found in XR), which forms a hydrogen bond to the proton acceptor from the same molecule (Asp97) and also to Trp34 of a neighboring protomer. Trp34 may function by stabilizing His75 in a conformation that favors a deprotonated Asp97 in the dark state, and suggests cooperative behavior between protomers when the protein is in an oligomeric form. Mutation-induced alterations in proton transfers in the BPR photocycle in Escherichia coli cells provide evidence for a similar cross-protomer interaction of BPR in living cells and a functional role of the inter-protomer Trp34-His75 interaction in ion transport. Finally, Wat402, a key molecule responsible for proton translocation between the Schiff base and the proton acceptor in BR, appears to be absent in PR, suggesting that the ion-transfer mechanism may differ between PR and BR.

Comparative genome analyses of Serratia marcescens FS14 reveals its high antagonistic potential.

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.

Crystallization and preliminary X-ray crystallographic analysis of a blue-light-absorbing proteorhodopsin

Proteorhodopsins (PRs), seven-transmembrane chromoproteins with retinal as a chromophore, are light-driven proton pumps. To elucidate the light-driven proton-pumping mechanism of PRs, a pET28a vector containing the blue-light-absorbing proteorhodopsin (BPR) gene was constructed and the protein was overexpressed in Escherichia coli. The protein was purified by immobilized metal-ion affinity chromatography (IMAC). The purified BPR D97N mutant protein (BPR_D97N) was crystallized using the vapour-diffusion method. Preliminary X-ray diffraction data analysis showed that the crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 161.6, b = 168.6, c = 64.7 Å. A complete data set was collected to 3.3 Å resolution using synchrotron radiation on beamline X06 of the Swiss Light Source (SLS). Molecular replacement was unsuccessful. To solve the structure of BPR_D97N by experimental phasing, selenomethionine-substituted protein crystals were prepared. These crystals diffracted to 3.0 Å resolution and a complete data set was collected on beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF). Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contained five molecules in the asymmetric unit, with a V(M) of 3.26 Å(3) Da(-1) and a solvent content of 62.3%.

Isolation of proteorhodopsin-bearing bacterium JL-3 from fresh water and characterization of the proteorhodopsin

Proteorhodopsins (PRs), light-driven proton pumps, constitute the largest family of the microbial rhodopsins. PRs are widely distributed in the oceanic environment and freshwater, but no bacteria with PRs have been isolated from freshwater so far. To facilitate isolation of the bacteria with PR genes, we constructed a vector system that can be used to clone potential PR genes and render color changes when overexpressed in Escherichia coli. Using this method, we successfully isolated a strain with PR gene from freshwater and identified it as Exiguobacterium sp. JL-3. The full length PR gene was then cloned using the SEFA PCR method. Protein sequence alignment showed that JL-3_PR shares high sequence identity (84-89%) with the PRs from Exiguobacterium strains, but low sequence identity (< 38%) with other PRs. Surprisingly, we could not detect any proton-pumping activity in the native JL-3 cells and protoplasts, but the recombinant JL-3_PR do pump protons when overexpressed in E. coli. Sequence analysis further revealed that the PRs from Exiguobacterium had an unusual lysine as the proton donor instead of the typical acidic residue. These data suggest that JL-3_PR is a sensory PR rather than a proton pump.

Identification of a toxic serralysin family protease with unique thermostable property from S. marcescens FS14

A serralysin family protease (Serralysin-like protease B, SPB) with unique V-shaped thermostable property was isolated and identified from the Serratia marcescens FS14 by biochemical and molecular biological methods. It is the first time to report the isolation of a native serralysin family protease directly from Serratia species except the well-studied serralysin. SPB has an optimum pH at 8.0 and an optimum temperature at 37°C. It shows high proteolytic activities after pretreated at 4-50°C for 10min respectively and almost no detectable activity after pretreated at 60°C. Surprisingly, increasing activities were observed after pretreated at 70-90°C respectively. Further study revealed that the reason behind this phenomenon may be the self-digestion property of SPB with an optimum temperature around 60°C. This self-digestion property may expand the SPB future application in industry. The bioassay using the healthy cotton bollworm Helicoverpa armigera larvae demonstrated that the serralysin and SPB from FS14 are toxic to the H. armigera larvae. This result implied that FS14 strain and/or the SPB and serralysin in FS14 might have a potential application in insect control.

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway.

Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N- and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ∼9400 Å3. The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases.

Expression, crystallization and preliminary crystallographic data analysis of VioD, a hydroxylase in the violacein-biosynthesis pathway.

Violacein, a natural purple secondary metabolite, is sequentially biosynthesized by five enzymes in the following pathway: VioA-VioB-VioE-VioD-VioC. VioD, a flavin-dependent oxygenase, catalyzes the hydroxylation of the intermediate product prodeoxyviolaceinic acid (PVA) at the 5-position of one indole ring to yield proviolacein. In vitro biochemical data have revealed this process, but the catalytic mechanism still remains largely unclear. Here, the cloning, expression, purification, crystallization and diffraction of VioD are reported. Crystals of VioD diffracted to 1.7 Å resolution and belonged to space group P31, with unit-cell parameters a = b = 90.0, c = 94.5 Å, α = β = 90, γ = 120°. Solvent-content calculation and molecular-replacement results suggest the presence of two molecules of VioD in the asymmetric unit.

Expression, purification, crystallization and preliminary crystallographic analysis of Cg1458: a novel oxaloacetate decarboxylase from Corynebacterium glutamicum.

Oxaloacetate decarboxylase catalyses the decarboxylation of oxaloacetate to pyruvate and CO(2). Recently, the Corynebacterium glutamicum gene product Cg1458 was determined to be a soluble oxaloacetate decarboxylase. To elucidate the mechanism of oxaloacetate decarboxylation by Cg1458, recombinant Cg1458 was purified and crystallized. The best crystal was grown from 0.2 M MgCl(2), 0.1 M Bis-Tris pH 6.0, 25%(w/v) polyethylene glycol 3350 using the hanging-drop method. The crystals belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 124.1, c = 73.6 Å. The crystals are most likely to contain a dimer in the asymmetric unit, with a V(M) value of 2.27 Å(3) Da(-1). A full data set was collected at 1.9 Å resolution using synchrotron radiation on beamline BL17U of SSRF, Shanghai, China. Structure-solution attempts by molecular replacement were successful with PDB entries 3qdf or 2dfu as the template.

Crystal structures of the kinase domain of PpkA, a key regulatory component of T6SS, reveal a general inhibitory mechanism

The type VI secretion system (T6SS) is a versatile and widespread export system found in many Gram-negative bacteria that delivers effector proteins into target cells. The functions of T6SSs are tightly regulated by diverse mechanisms at multiple levels, including post-translational modification through threonine phosphorylation via the Ser/Thr protein kinase (STPK) PpkA. Here, we identified that PpkA is essential for T6SS secretion in Serratia marcescens since its deletion eliminated the secretion of haemolysin co-regulated protein, while the periplasmic and transmembrane portion of PpkA was found to be disposable for T6SS secretion. We further determined the crystal structure of the kinase domain of PpkA (PpkA-294). The structure of PpkA-294 was determined in its apo form to a 1.6 Å resolution as well as in complex with ATP to a 1.41 Å resolution and with an ATP analogue AMP-PCP to a 1.45 Å resolution. The residues in the activation loop of PpkA-294 were fully determined, and the N-terminus of the loop was folded into an unprecedented inhibitory helix, revealing that the PpkA kinase domain was in an auto-inhibitory state. The ternary MgATP-PpkA-294 complex was also inactive with nucleotide ribose and phosphates in unexpected and unproductive conformations. The αC-helix in the inactive PpkA-294 adopted a conformation towards the active site but with the conserved glutamate in the helix rotated away, which we suggest to be a general conformation for all STPK kinases in the inactive form. Structural comparison of PpkA with its eukaryotic homologues reinforced the universal regulation mechanism of protein kinases.

Myroilysin Is a New Bacterial Member of the M12A Family of Metzincin Metallopeptidases and Is Activated by a Cysteine Switch Mechanism

Proteases play important roles in all living organisms and also have important industrial applications. Family M12A metalloproteases, mainly found throughout the animal kingdom, belong to the metzincin protease family and are synthesized as inactive precursors. So far, only flavastacin and myroilysin, isolated from bacteria, were reported to be M12A proteases, whereas the classification of myroilysin is still unclear due to the lack of structural information. Here, we report the crystal structures of pro-myroilysin from bacterium Myroides sp. cslb8. The catalytic zinc ion of pro-myroilysin, at the bottom of a deep active site, is coordinated by three histidine residues in the conserved motif HEXXHXXGXXH; the cysteine residue in the pro-peptide coordinates the catalytic zinc ion and inhibits myroilysin activity. Structure comparisons revealed that myroilysin shares high similarity with the members of the M12A, M10A, and M10B families of metalloproteases. However, a unique “cap” structure tops the active site cleft in the structure of pro-myroilysin, and this “cap” structure does not exist in the above structure-reported subfamilies. Further structure-based sequence analysis revealed that myroilysin appears to belong to the M12A family, but pro-myroilysin uses a “cysteine switch” activation mechanism with a unique segment, including the conserved cysteine residue, whereas other reported M12A family proteases use an “aspartate switch” activation mechanism. Thus, our results suggest that myroilysin is a new bacterial member of the M12A family with an exceptional cysteine switch activation mechanism. Our results shed new light on the classification of the M12A family and may suggest a divergent evolution of the M12 family.