Molecular mechanism governing RNA-binding property of mammalian TRIM71 protein.

TRIM71 is an RNA-binding protein with ubiquitin ligase activity. Numerous functions of mammalian TRIM71, including cell cycle regulation, embryonic stem cell (ESC) self-renewal, and reprogramming of pluripotent stem cells, are related to its RNA-binding property. We previously reported that a long noncoding RNA (lncRNA) Trincr1 interacts with mouse TRIM71 (mTRIM71) to repress FGF/ERK pathway in mouse ESCs (mESCs). Herein, we identify an RNA motif specifically recognized by mTRIM71 from Trincr1 RNA, and solve the crystal structure of the NHL domain of mTRIM71 complexed with the RNA motif. Similar to the zebrafish TRIM71, mTRIM71 binds to a stem-loop structured RNA fragment of Trincr1, and an adenosine base at the loop region is crucial for the mTRIM71 interaction. We map similar hairpin RNAs preferably bound by TRIM71 in the mRNA UTRs of the cell-cycle related genes regulated by TRIM71. Furthermore, we identify key residues of mTRIM71, conserved among mammalian TRIM71 proteins, required for the RNA-binding property. Single-site mutations of these residues significantly impair the binding of TRIM71 to hairpin RNAs in vitro and to mRNAs of Cdkn1a/p21 and Rbl2/p130 in mESCs. Furthermore, congenital hydrocephalus (CH) specific mutation of mTRIM71 impair its binding to the RNA targets as well. These results reveal molecular mechanism behind the recognition of RNA by mammalian TRIM71 and provide insights into TRIM71 related diseases.

Structural characterization of fungus-specific histone deacetylase Hos3 provides insights into developing selective inhibitors with antifungal activity.

Fungal infection has long been a chronic and even life-threatening problem for humans. The demand for new antifungal drugs has increased dramatically as fungal infections have continued to increase, yet no new classes of drugs have been approved for nearly 15 years due to either high toxicity or development of drug resistance. Thus, validating new drug targets, especially fungus-specific targets, may facilitate future drug design. Here, we report the crystal structure of yeast Hos3 (ScHos3), a fungus-specific histone deacetylase (HDAC) that plays an important role in the life span of fungi. As acetylation modifications are important to many aspects of fungal infection, the species specificity of Hos3 makes it an ideal target for the development of new antifungal drugs. In this study, we show that ScHos3 forms a functional homodimer in solution, and key residues for dimerization crucial for its deacetylation activity were identified. We used molecular dynamics simulation and structural comparison with mammalian hHDAC6 to determine unique features of the ScHos3 catalytic core. In addition, a small-molecule inhibitor with a preference for ScHos3 was identified through structure-based virtual screening and in vitro enzymatic assays. The structural information and regulatory interferences of ScHos3 reported here provide new insights for the design of selective inhibitors that target fungal HDAC with high efficiency and low toxicity or that have the potential to overcome the prevailing problem of drug resistance in combination therapy with other drugs.

Structural characterization of the urease accessory protein UreF from Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that mostly affects those with weakened immune systems. Urease is a vital enzyme that can hydrolyze urea to ammonia and carbon dioxide as a source of nitrogen for growth. Urease is also a K. pneumoniae virulence factor that enables survival of the bacterium under nutrient-limiting conditions. UreF, an important nickel-binding urease accessory protein, is involved in the insertion of Ni2+ into the active site of urease. Here, the crystal structure of UreF from K. pneumoniae (KpUreF) is reported. Functional data show that KpUreF forms a stable dimer in solution. These results may provide a starting point for the design of urease inhibitors.

Structure of Pseudomonas aeruginosa spermidine dehydrogenase: a polyamine oxidase with a novel heme-binding fold.

The opportunistic pathogen Pseudomonas aeruginosa can utilize polyamines (including putrescine, cadaverine, 4-aminobutyrate, spermidine, and spermine) as its sole source of carbon and nitrogen. Spermidine dehydrogenase (SpdH) is a component of one of the two polyamine utilization pathways identified in P. aeruginosa, but little is known about its structure and function. Here, we report the first crystal structure of SpdH from P. aeruginosa to 1.85 Å resolution. The resulting core structure confirms that SpdH belongs to the polyamine oxidase (PAO) family with flavin-binding and substrate-binding domains. A unique N-terminal extension wraps around the flavin-binding domain of SpdH and is required for heme binding, placing a heme cofactor in close proximity to the FAD cofactor. Structural and mutational analysis reveals that residues in the putative active site at the re side of the FAD isoalloxazine ring form part of the catalytic machinery. PaSpdH features an unusual active site and lacks the conserved lysine that forms part of a lysine-water-flavin N5 atom interaction in other PAO enzymes characterized to date. Mutational analysis further confirms that heme is required for catalytic activity. This work provides an important starting point for understanding the role of SpdH, which occurs universally in P. aeruginosa strains, in polyamine metabolism.

PHF8-promoted TOPBP1 demethylation drives ATR activation and preserves genome stability.

The checkpoint kinase ATR [ATM (ataxia-telangiectasia mutated) and rad3-related] is a master regulator of DNA damage response. Yet, how ATR activity is regulated remains to be investigated. We report here that histone demethylase PHF8 (plant homeodomain finger protein 8) plays a key role in ATR activation and replication stress response. Mechanistically, PHF8 interacts with and demethylates TOPBP1 (DNA topoisomerase 2-binding protein 1), an essential allosteric activator of ATR, under unperturbed conditions, but replication stress results in PHF8 phosphorylation and dissociation from TOPBP1. Consequently, hypomethylated TOPBP1 facilitates RAD9 (RADiation sensitive 9) binding and chromatin loading of the TOPBP1-RAD9 complex to fully activate ATR and thus safeguard the genome and protect cells against replication stress. Our study uncovers a demethylation and phosphorylation code that controls the assembly of TOPBP1-scaffolded protein complex, and provides molecular insight into non-histone methylation switch in ATR activation.

Structure of subunit I of the anthranilate synthase complex of Mycolicibacterium smegmatis.

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.

Structural characterization of the Pseudomonas aeruginosa dehydrogenase AtuB involved in citronellol and geraniol catabolism.

Pseudomonas aeruginosa can metabolize acyclic monoterpenoids (such as citronellol and geraniol) as the only carbon and energy sources. A total of seven proteins (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG, AtuH) have been identified in Pseudomonas aeruginosa as participating in the acyclic terpene utilization pathway. AtuB is a dehydrogenase enzyme responsible for citronellol and geraniol catabolism in the acyclic terpene utilization (Atu) pathway, although its structure and function have not been characterized to date. Here we report the crystal structure of AtuB from Pseudomonas aeruginosa PAO1 (PaAtuB) to 1.8 Å resolution. PaAtuB crystallizes in the space group F222 with a single monomer in the asymmetric unit. Analytical ultracentrifugation data shows that PaAtuB forms a stable tetramer in solution, which is consistent with the structure. Structural analysis confirms that AtuB belongs to the short-chain dehydrogenase/reductase (SDR) family. AtuB is predicted to bind NADP(H) from the crystal structure, which is confirmed by MicroScale Thermophoresis analysis that shows PaAtuB binds NADP(H) with a Kd value of 258 µM. This work provides a starting point to explore potential biotechnology and pharmaceutical applications of AtuB.

Universal stress protein in Malus sieversii confers enhanced drought tolerance.

Drought is an important environmental factor that can severely affect plant growth and reproduction. Although many genes related to drought tolerance have been studied in economically important crops, very few genes have been functionally identified in Malus sieversii. In this study, we isolated a new gene based on throughput RNA sequencing analysis and constructed genetic expression vectors and transformed in Arabidopsis thaliana for functional verification. The results showed that MsUspA ectopic expression driven by constitutive (CaMV 35S) promoter gave rise to substantial improvements in ability of transgenic A. thaliana plants to survive under extreme drought conditions. Improved drought resistance mainly depends on more compact cellular structure, longer roots, strong resilience and low-level ROS. Molecular expression analysis showed that MsUspA may be involved in hormone and secondary metabolite synthesis regulation to improve drought resistance.

Structural characterization of an isopenicillin N synthase family oxygenase from Pseudomonas aeruginosa PAO1.

Isopenicillin N synthase (IPNS) is a nonheme-Fe2+-dependent enzyme that mediates a key step in penicillin biosynthesis. It catalyses the conversion of the tripeptide δ-(l-α-aminoadipoyl)-l-cysteine-d-valine (ACV) to isopenicillin N, which is a key precursor to ß-lactam antibiotics. The pa4191 gene in Pseudomonas aeruginosa PAO1 has provisionally been annotated as a member of the IPNS family. In this work, we report the crystal structure of PA4191 from P. aeruginosa (PaIPNS hereafter). The 1.65 Šresolution PaIPNS structure forms a jelly roll fold and is confirmed to be a member of the IPNS family based on structural homology. A metal centre within the jelly roll consists of the strictly conserved His201, Asp203 and His257 residues. MicroScale Thermophoresis binding analysis confirms that PaIPNS is a metal-binding protein with a strong preference for iron, but that it does not bind the tripeptide ACV. Structural comparison of PaIPNS with a previously reported IPNS-ACV complex structure reveals a restricted binding pocket that is unable to accommodate ACV.

Structural characterization of an acetolactate decarboxylase from Klebsiella pneumoniae.

Acetolactate decarboxylase (ALDC) is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin), an important physiological metabolite excreted by microbes. Although the enzyme is widely present in microorganisms, few atomic structures and functions of ALDC have been reported to date. Here we report the crystal structure of ALDC from Klebsiella pneumoniae KP (KpALDC). KpALDC crystallizes in space group P3121 with one monomer per asymmetric unit. Analytical ultracentrifugation data shows that KpALDC forms a stable dimer but can exist as a tetramer in solution. A Zn2+ ion is coordinated by three strictly-conserved histidines (His198, His200 and His211) and a conserved glutamate (Glu69), but the C-terminal tail that forms part of the active site in ALDC enzymes is disordered. A complex structure with ethane-1,2-diol shows a unusual mode of binding, whereby the ligand does not coordinate the Zn2+ ion. MicroScale Thermophoresis analysis shows that KpALDC binds Zn2+ ions, but no binding of Mg2+, Ca2+ and Mn2+ ions was detected.

Crystal structure of a glutamate-1-semialdehyde-aminomutase from Pseudomonas aeruginosa PAO1.

The C5 pathway in bacteria is responsible for the synthesis of 5-aminolevulinic acid, which forms the core skeleton of cofactors required for metabolism. One of the key actors in this pathway is a pyridoxamine-5'-phosphate (PMP)/pyridoxal-5'-phosphate (PLP) dependent enzyme called glutamate-1-semialdehyde aminomutase (GSAM). In this study, we crystallized the expression product of the uncharacterized pa4088 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The resulting high-resolution structure confirms it to be a member of the GSAM family. Continuous electron density indicates the presence of a PLP cofactor with a Schiff base linkage between the PLP cofactor and the ε-amino group of Lys286. A crystal structure of a K286A mutant in complex with PMP is also reported. As GSAM enzymes are not present in mammalian cells, this work provides a starting point for the investigation of GSAM as a target for drug development against P. aeruginosa infection.

Crystal structure of RecR, a member of the RecFOR DNA-repair pathway, from Pseudomonas aeruginosa PAO1.

DNA damage is usually lethal to all organisms. Homologous recombination plays an important role in the DNA damage-repair process in prokaryotic organisms. Two pathways are responsible for homologous recombination in Pseudomonas aeruginosa: the RecBCD pathway and the RecFOR pathway. RecR is an important regulator in the RecFOR homologous recombination pathway in P. aeruginosa. It forms complexes with RecF and RecO that can facilitate the loading of RecA onto ssDNA in the RecFOR pathway. Here, the crystal structure of RecR from P. aeruginosa PAO1 (PaRecR) is reported. PaRecR crystallizes in space group P6122, with two monomers per asymmetric unit. Analytical ultracentrifugation data show that PaRecR forms a stable dimer, but can exist as a tetramer in solution. The crystal structure shows that dimeric PaRecR forms a ring-like tetramer architecture via crystal symmetry. The presence of a ligand in the Walker B motif of one RecR subunit suggests a putative nucleotide-binding site.

Identification of MsHsp20 Gene Family in Malus sieversii and Functional Characterization of MsHsp16.9 in Heat Tolerance.

Heat shock proteins (Hsps) are common molecular chaperones present in all plants that accumulate in response to abiotic stress. Small heat shock proteins (sHsps) play important roles in alleviating diverse abiotic stresses, especially heat stress. However, very little is known about the MsHsp20 gene family in the wild apple Malus sieversii, a precious germplasm resource with excellent resistance characteristics. In this study, 12 putative M. sieversii Hsp20 genes were identified from RNA-Seq data and analyzed in terms of gene structure and phylogenetic relationships. A new Hsp20 gene, MsHsp16.9, was cloned and its function studied in response to stress. MsHsp16.9 expression was strongly induced by heat, and transgenic Arabidopsis plants overexpressing MsHsp16.9 displayed improved heat resistance, enhanced antioxidant enzyme activity, and decreased peroxide content. Overexpression of MsHsp16.9 did not alter the growth or development under normal conditions, or the hypersensitivity to exogenous ABA. Gene expression analysis indicated that MsHsp16.9 mainly modulates the expression of proteins involved in antioxidant enzyme synthesis, as well as ABA-independent stress signaling in 35S:MsHsp16.9-L11. However, MsHsp16.9 could activate ABA-dependent signaling pathways in all transgenic plants. Additionally, MsHsp16.9 may function alongside AtHsp70 to maintain protein homeostasis and protect against cell damage. Our results suggest that MsHsp16.9 is a protein chaperone that positively regulates antioxidant enzyme activity and ABA-dependent and independent signaling pathway to attenuate plant responses to severe stress. Transgenic plants exhibited luxuriant growth in high temperature environments.

Structural insights into YfiR sequestering by YfiB in Pseudomonas aeruginosa PAO1.

YfiBNR is a tripartite signalling system in Pseudomonas aeruginosa that modulates intracellular c-di-GMP levels in response to signals received in the periplasm. YfiB is an outer membrane lipoprotein and presumed sensor protein that sequesters the repressor protein YfiR. To provide insights into YfiBNR function, we have determined three-dimensional crystal structures of YfiB and YfiR from P. aeruginosa PAO1 alone and as a 1:1 complex. A YfiB(27-168) construct is predominantly dimeric, whereas a YfiB(59-168) is monomeric, indicating that YfiB can dimerize via its N-terminal region. YfiR forms a stable complex with YfiB(59-168), while the YfiR binding interface is obstructed by the N-terminal region in YfiB(27-168). The YfiB-YfiR complex reveals a conserved interaction surface on YfiR that overlaps with residues predicted to interact with the periplasmic PAS domain of YfiN. Comparison of native and YfiR-bound structures of YfiB suggests unwinding of the N-terminal linker region for attachment to the outer membrane. A model is thus proposed for YfiR sequestration at the outer membrane by YfiB. Our work provides the first detailed insights into the interaction between YfiB and YfiR at the molecular level and is a valuable starting point for further functional and mechanistic studies of the YfiBNR signalling system.