The DJ-1 superfamily (DJ-1/ThiJ/PfpI superfamily) is distributed across all three kingdoms of life. These proteins are involved in a highly diverse range of cellular functions, including chaperone and protease activity. DJ-1 proteins usually form dimers or hexamers in vivo and show at least four different binding orientations via distinct interface patches. Abnormal oligomerization of human DJ-1 is related to neurodegenerative disorders including Parkinson’s disease, suggesting important functional roles of quaternary structures. However, the quaternary structures of the DJ-1 superfamily have not been extensively studied. Here, we focus on the diverse oligomerization modes among the DJ-1 superfamily proteins and investigate the functional roles of quaternary structures both computationally and experimentally. The oligomerization modes are classified into 4 types (DJ-1, YhbO, Hsp, and YDR types) depending on the distinct interface patches (I–IV) upon dimerization. A unique, rotated interface via patch I is reported, which may potentially be related to higher order oligomerization. In general, the groups based on sequence similarity are consistent with the quaternary structural classes, but their biochemical functions cannot be directly inferred using sequence information alone. The observed phyletic pattern suggests the dynamic nature of quaternary structures in the course of evolution. The amino acid residues at the interfaces tend to show lower mutation rates than those of non-interfacial surfaces.
DJ-1 superfamily; DJ-1/ThiJ/PfpI superfamily; quaternary structure
Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.
Legionella pneumophila is a pathogen bacterium that causes Legionnaires' disease accompanied by severe pneumonia. Surprisingly, this pathogen invades and replicates inside macrophages, whose major function is to detect and destroy invading microorganisms. How L. pneumophila can be “immune” to this primary immune cell has been a focus of intensive research. Upon being engulfed by a macrophage cell, L. pneumophila translocates hundreds of bacterial proteins into this host cell. These proteins, called bacterial effectors, are thought to manipulate normal host cellular processes. However, which host molecules and how they are targeted by the bacterial effectors are largely unknown. In this study, we determined the three-dimensional structure of L. pneumophila effector protein VipD, whose function in macrophage was unknown. Ensuing analyses revealed that VipD selectively and tightly binds two host signaling proteins Rab5 and Rab22, which are key regulators of early endosomal vesicle trafficking. These interactions prevent the activated form of Rab5 and Rab22 from binding their downstream signaling proteins, resulting in the blockade of endosomal trafficking in macrophages. The presented work shows that L. pneumophila targets endosomal Rab proteins and delineates the underlying molecular mechanism, providing a new insight into the pathogen's strategies to dysregulate normal intracellular processes.
A recombinant multiple cofactor-dependent DNA ligase from S. zilligii has been purified and crystallized. X-ray diffraction data were collected to 2.9 Å resolution and the crystals belonged to space group P1.
A recombinant DNA ligase from Sulfophobococcus zilligii that shows multiple cofactor specificity (ATP, ADP and GTP) was expressed in Escherichia coli and purified under reducing conditions. Crystals were obtained by the microbatch crystallization method at 295 K in a drop containing 1 µl protein solution (10 mg ml−1) and an equal volume of mother liquor [0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 10 000]. A data set was collected to 2.9 Å resolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a = 63.7, b = 77.1, c = 77.8 Å, α = 83.4, β = 82.4, γ = 74.6°. Assuming the presence of two molecules in the unit cell, the solvent content was estimated to be about 53.4%.
multiple cofactors; DNA ligases; Sulfophobococcus zilligii
The surface of proteins can be charged with zinc ions and the anomalous signals from these zinc ions can be used for structure determination of proteins.
Zinc is a suitable metal for anomalous dispersion phasing methods in protein crystallography. Structure determination using zinc anomalous scattering has been almost exclusively limited to proteins with intrinsically bound zinc(s). Here, it is reported that multiple zinc ions can easily be charged onto the surface of proteins with no intrinsic zinc-binding site by using zinc-containing solutions. Zn derivatization of protein surfaces appears to be a largely unnoticed but promising method of protein structure determination.
zinc anomalous scattering; phasing; Zn derivatization
It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various β-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.
Lon is an oligomeric ATP-dependent protease that degrades defective or denatured proteins as well as some folded proteins for the control of cellular protein quality and metabolism. Lon from T. onnurineus NA1 has been purified and crystallized at 295 K.
Lon is an oligomeric ATP-dependent protease that degrades defective or denatured proteins as well as some folded proteins for the control of cellular protein quality and metabolism. Lon from Thermococcus onnurineus NA1 was purified and crystallized at 295 K. A 2.0 Å resolution data set was collected using synchrotron radiation. The crystals belonged to space group P63, with unit-cell parameters a = 121.45, b = 121.45, c = 195.24 Å. Assuming the presence of two monomers in the asymmetric unit, the solvent content was estimated to be about 60.7%.
Lon; ATP-dependent proteases; Thermococcus onnurineus NA1
The TON_0887 gene product was purified and crystallized at 295 K. A 2.2 Å resolution data set was collected using synchrotron radiation.
The TON_0887 gene product from Thermococcus onnurineus NA1 is a 240-residue protein that has histidinol-phosphate phosphatase (HolPase) activity. According to analysis of its primary structure, the TON_0887 gene product is a monofunctional HolPase that belongs to the DDDD superfamily. This contrasts with the generally accepted classification that bifunctional HolPases belong to the DDDD superfamily. The TON_0887 gene product was purified and crystallized at 295 K. A 2.2 Å resolution data set was collected using synchrotron radiation. The TON-HolPase crystals belonged to space group P2221, with unit-cell parameters a = 40.88, b = 46.89, c = 148.03 Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 48.3%.
histidinol-phosphate phosphatase; Thermococcus onnurineus NA1
A nickel uptake regulator (Nur) from Streptomyces coelicolor has been crystallized. A 2.4 Å native data set and a 3.0 Å Ni-MAD data set were collected using synchrotron radiation.
Nickel ions serve in the correct folding and function of microbial enzymes implicated in metabolic processes. Although nickel ions are indispensable for the survival of cells, the intracellular level of nickel ions needs to be properly maintained as excessive levels of nickel ions are toxic. Nur, a nickel-uptake regulator belonging to the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and antioxidative response in Streptomyces coelicolor. Nur was purified and crystallized at 295 K. A 2.4 Å native data set and a 3.0 Å Ni-MAD data set were collected using synchrotron radiation. The Nur crystals belong to space group P31, with unit-cell parameters a = b = 78.17, c = 50.39 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is estimated to be about 54.7%.
Nur; nickel; transcription factors; Streptomyces coelicolor
Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO2, thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.
Nur, a member of the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and anti-oxidative response in Streptomyces coelicolor. Here we report the 2.4-Å resolution crystal structure of Nur. It contains a unique nickel-specific metal site in addition to a nonspecific common metal site. The identification of the 6-5-6 motif of the Nur recognition box and a Nur/DNA complex model reveals that Nur mainly interacts with terminal bases of the palindrome on complex formation. This contrasts with more distributed contacts between Fur and the n-1-n type of the Fur-binding motif. The disparity between Nur and Fur in the conformation of the S1-S2 sheet in the DNA-binding domain can explain their different DNA-recognition patterns. Furthermore, the fact that the specificity of Nur in metal sensing and DNA recognition is conferred by the specific metal site suggests that its introduction drives the evolution of Nur orthologs in the Fur family.
Maize ribosome-inactivating protein is classified as a class III or an atypical RNA N-glycosidase. It is synthesized as an inactive precursor with a 25-amino acid internal inactivation region, which is removed in the active form. As the first structural example of this class of proteins, crystals of the precursor and the active form were diffracted to 2.4 and 2.5 Å, respectively. The two proteins are similar, with main chain root mean square deviation (RMSD) of 0.519. In the precursor, the inactivation region is found on the protein surface and consists of a flexible loop followed by a long α-helix. This region diminished both the interaction with ribosome and cytotoxicity, but not cellular uptake. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure reveals that the active site is too small to accommodate two glutamate residues. Our structure suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins.
Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1.
The 1.6 Å crystal structure of PedB reveals that PedB consists of an antiparallel four-helix bundle with a flexible C-terminal end. PedB shows structural similarity to an immunity protein against enterocin A (EntA-im) but some disparity to an immunity protein against carnobacteriocin B2 (ImB2) in both the C-terminal conformation and the local structure constructed by α3, α4, and their connecting loop. Structure-inspired mutational studies reveal that deletion of the last seven residues of the C-terminus of PedB almost abolished its immunity activity.
The fact that PedB, EntA-im, and ImB2 share a four-helix bundle structure strongly suggests the structural conservation of this motif in the pediocin-like immunity proteins. The significant difference in the core structure and the C-terminal conformation provides a structural basis for the classification of pediocin-like immunity proteins. Our mutational study using C-terminal-shortened PedBs and the investigation of primary sequence of the C-terminal region, propose that several polar or charged residues in the extreme C-terminus of PedB which is crucial for the immunity are involved in the specific recognition of pediocin PP-1.