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Catechol‐O‐methyltransferase (COMT1) catalyzes the transfer of a methyl group from S‐adenosylmethionine (SAM) to various catechol substrates. COMTs play vital roles in physiological processes in animals, plants, and fungi, as well as bacteria, and have essential application values in industry. spCOMT is a probable COMT from Schizosaccharomyces pombe. It has an extraordinary intracellular distribution different from other homologs and would thus be predicted to perform a distinct physiological function. In this report, recombinant spCOMT was purified and kinetically characterized for the first time. The enzymology assays indicate that spCOMT is a metal‐dependent enzyme and belongs to class I OMTs. In addition, the crystal structures of apo‐spCOMT and SAM‐complexed spCOMT were also presented, revealing that spCOMT possesses a conserved SAM‐binding site and Mg2+ pocket, but a distinct substrate pocket was not present in homologs. The mutagenesis ITC analysis revealed the SAM recognition characteristics of spCOMT. Based on all of the above findings, we speculated about the putative substrates' characteristics and the substrate recognition mechanisms of spCOMT. This work will help in elucidating the physiological functions of spCOMT in S. pombe. © 2018 IUBMB Life, 71(3):330–339, 2019 [ABSTRACT FROM AUTHOR]

The lack of incorporating epitope information into the selection process makes the conventional antibody screening method less effective in identifying antibodies with desired functions. Here, we developed an epitope-directed antibody selection method by designing a directed library favoring the target epitope and a precise "counter" antigen for clearing irrelevant binders in the library. With this method, we successfully isolated an antibody, pF7_A5, that targets the less conserved region on the FZD2/7 CRD as designed. Guided by the structure of pF7_A5-FZD2CRD, a further round of evolution was conducted together with the "counter" antigen selection strategy, and ultimately, an FZD2-specific antibody and an FZD7-preferred antibody were obtained. Because of targeting the predefined functional site, all these antibodies exhibited the expected modulatory activity on the Wnt pathway. Together, the method developed here will be useful in antibody drug discovery, and the identified FZD antibodies will have clinical potential in FZD-related cancer therapy. [Display omitted] • Epitope-directed antibody identification aided by library and antigen design • An antibody targeting the desired Frizzled 2/7 site II region was identified • Structure-guided evolution enables the identification of highly specific variants • Epitope-specific antibodies show the expected modulations of the Wnt pathway The identification of antibodies with desired modulatory activities remains a challenging task. Ge et al. established a broadly applicable antibody isolation method that allows the selection of antibodies targeting specific regions on Frizzled receptors. The isolated antibodies showed the expected modulations of the Frizzled receptor-mediated signaling pathway. [ABSTRACT FROM AUTHOR]

Examines the crystal structure of haemorrhagins (AaH I), and determine the relationship between AaH I and matrix matalloproteinases (MMP) super-family based on their structural characteristics. Where the Agkistrodon acutus snake venom was collected from; Indication that AaH I was purified and crystallized; How the crystal structure of AaH I was determined; Results and discussion on the study.

The Saccharomyces cerevisiae NAD+-dependent deacetylase HST1 belongs to the class III HDAC family; it acts as a transcriptional corepressor for the specific middle sporulation and de novo NAD+-biosynthesis genes and also takes part in the SET3C and SUM1-RFM1-HST1 complexes. Structural information on HST1 and its related complexes would be helpful in order to understand the structural basis of its deacetylation mechanism and the assembly of these complexes. Here, HST1156-503 was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.90 Å resolution and belonged to space group P21, with unit-cell parameters a = 40.2, b = 101.7, c = 43.9 Å, β = 103.9°. Both Matthews coefficient analysis and the self-rotation function suggested the presence of four molecules per asymmetric unit in the crystal, with a solvent content of 49.76% ( VM = 2.45 Å3 Da−1). [ABSTRACT FROM AUTHOR]

Cyclobutane pyrimidine dimers (CPD), as a common DNA damage caused by UV radiation, often lead to skin cancer. Here, we identified a photolyase from the alga Arthrospira platensis (designated as Ap‐phr), which has been regarded as a safe organism for humans for centuries, that can efficiently repair CPD lesions in ssDNA and dsDNA in vitro. The 1.6 Å resolution crystal structure of Ap‐phr revealed that it possesses a unique methenyltetrahydrofolate chromophore‐binding pattern with high energy transfer efficiency. Our study of Ap‐phr highlights its potential use in cosmetic, industrial and aesthetic medicine applications. [ABSTRACT FROM AUTHOR]

Intracellular fatty acid-binding proteins are evolutionarily highly conserved proteins. The major functions and responsibilities of this family are the regulation of FA uptake and intracellular transport. The structure of the H-FABP ortholog from mouse (Mus musculus) had not been revealed at the time this study was completed. Thus, further exploration of the structural properties of mouse H-FABP is expected to extend our knowledge of the model animal's molecular mechanism of H-FABP function. Here, we report the high-resolution crystal structure and the NMR characterization of mouse H-FABP. Our work discloses the unique structural features of mouse H-FABP, offering a structural basis for the further development of small-molecule inhibitors for H-FABP. [ABSTRACT FROM AUTHOR]

RsmA, a ribosomal RNA small subunit methyltransferase from Staphylococcus aureus, catalyzes the N6 methylation of adenine in 16S rRNA. In this study, RsmA from Staphylococcus aureus was cloned, expressed, purified and crystallized. The crystal belonged to space group C2, with unit-cell parameters a = 84.38, b = 157.76, c = 96.50 Å, β = 95.04°. X-ray diffraction data were collected to a resolution of 3.2 Å. The self-rotation function and the Matthews coefficient suggested the presence of two molecules in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Abstract: We investigated what roles coevolution plays in shaping yeast protein interaction network (PIN). We found that the extent of coevolution between two proteins decreases rapidly as their interacting distance on the PIN increases, suggesting coevolutionary constraint is a short-distance force at the molecular level. We also found that protein–protein interactions (PPIs) with strong coevolution tend to be enriched in interconnected clusters, whereas PPIs with weak coevolution are more frequently present at inter-cluster region. The findings indicate the close relationship between coevolution and modular organization of PINs, and may provide insights into evolution and modularity of cellular networks. [ABSTRACT FROM AUTHOR]

Background: Recent progresses in high-throughput proteomics have provided us with a first chance to characterize protein interaction networks (PINs), but also raised new challenges in interpreting the accumulating data. Results: Motivated by the need of analyzing and interpreting the fast-growing data in the field of proteomics, we propose a comparative strategy to carry out global analysis of PINs. We compare two PINs by combining interaction topology and sequence similarity to identify conserved network substructures (CoNSs). Using this approach we perform twenty-one pairwise comparisons among the seven recently available PINs of E.coli, H.pylori, S.cerevisiae, C.elegans, D.melanogaster, M.musculus and H.sapiens. In spite of the incompleteness of data, PIN comparison discloses species conservation at the network level and the identified CoNSs are also functionally conserved and involve in basic cellular functions. We investigate the yeast CoNSs and find that many of them correspond to known complexes. We also find that different species harbor many conserved interaction regions that are topologically identical and these regions can constitute larger interaction regions that are topologically different but similar in framework. Based on the species-to-species difference in CoNSs, we infer potential species divergence. It seems that different species organize orthologs in similar but not necessarily the same topology to achieve similar or the same function. This attributes much to duplication and divergence of genes and their associated interactions. Finally, as the application of CoNSs, we predict 101 protein-protein interactions (PPIs), annotate 339 new protein functions and deduce 170 pairs of orthologs. Conclusion: Our result demonstrates that the cross-species comparison strategy we adopt is powerful for the exploration of biological problems from the perspective of networks. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a well‐known clinical pathogenic bacterium. In recent years, due to the emergence of multiple drug‐resistant strains of S. aureus in clinical practice, S. aureus infections have become an increasingly severe clinical problem. Ntdp (nucleoside tri‐ and diphosphatase, also known as Sa1684) is a nucleotide phosphatase that has a significant effect on the proliferation of S. aureus colonies and the killing ability of the host. Here, we identified the nucleoside tri‐ and diphosphate hydrolysis activity of Ntdp and obtained the three‐dimensional structures of apo‐Ntdp and three substrate analog (ATPγS, GDPβS, and GTPγS) complexes of Ntdp. Through structural analysis and biochemical verification, we illustrated the structural basis for the divalent cation selectivity, substrate recognition model, and catalytic mechanism of Ntdp. We also revealed a possible basal functional pattern of the DUF402 domain and hypothesized the potential pathways by which the protein regulates the expression of the two‐component regulatory factor agr and the downstream virulence factors. Overall, the above findings provide crucial insights into our understanding of the Ntdp functional mechanism in the infection process. [ABSTRACT FROM AUTHOR]

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation. Trial registration: Clinicaltrials.gov NCT01588132. [ABSTRACT FROM AUTHOR]

Adenylate kinase plays a very important role in regulating adenylate species in the cell. Methanocaldococcus jannaschii is a rich resource of unique enzymes. Here, MJ0458, an adenylate kinase from M. jannaschii, was crystallized. A set of X-ray diffraction data to 2.70 Å resolution was collected on beamline BL-17U of the Shanghai Synchrotron Radiation Facility (SSRF). The crystal belonged to space group P41212 or P43212. The unit-cell parameters were a = b = 76.18, c = 238.70 Å, α = β = γ = 90°. [ABSTRACT FROM AUTHOR]

• ArlRS is an essential two-component system in Staphylococcus aureus. • In this work, we identified different DNA binding properties and aggregation states among the DNA-binding domain of ArlR (ArlRDBD) and the phosphorylated and unphosphorylated full-length ArlR. We solved the crystal structure of ArlRDBD at 2.5-Å resolution and confirmed the DNA-binding site of ArlR and the preferred binding sequences in the agr promoter that enables the DNA recognition process. Finally, we propose a putative transcription regulation mechanism for ArlR. • This work will facilitate our understanding of the DNA binding affinity regulatory mechanism between the phosphorylated and unphosphorylated RR in TCSs. ArlRS is an essential two-component system in Staphylococcus aureus that regulates the transcription of virulence factors and participate in numerous pathogenic and symbiotic processes. In this work, we identified different DNA binding properties and oligomerization states among the DNA-binding domain of ArlR (ArlRDBD) and the phosphorylated and unphosphorylated full-length ArlR. Based on a 2.5-Å resolution crystal structure of ArlRDBD and subsequent mutagenesis experiments, we confirmed the DNA-binding site of ArlR and the preferred binding sequences in the agr promoter that enables the DNA recognition process. Finally, we propose a putative transcription regulation mechanism for ArlR. This work will facilitate our understanding of the DNA binding affinity regulatory mechanism between the phosphorylated and unphosphorylated response regulator in the two-component system. [ABSTRACT FROM AUTHOR]

The eubacterial β sliding clamp (DnaN) plays a crucial role in DNA metabolism through direct interactions with DNA, polymerases, and a variety of protein factors. A canonical protein–DnaN interaction has been identified in Escherichia coli and some other species, during which protein partners are tethered into the conserved canonical hydrophobic crevice of DnaN via the consensus β‐binding motif. Caulobacter crescentus is an excellent research model for use in the investigation of DNA replication and cell‐cycle regulation due to its unique asymmetric cell division pattern with restricted replication initiation; however, little is known about the specific features of C. crescentus DnaN (CcDnaN). Here, we report a significant divergence in the association of CcDnaN with proteins based on docking analysis and crystal structures that show that the β‐binding motifs of its protein partners bind a novel pocket instead of the canonical site. Pull‐down and isothermal titration calorimetry results revealed that mutations within the novel pocket disrupt protein–CcDnaN interactions. It was also shown by replication and regulatory inactivation of DnaA assays that mediation of protein interaction by the novel pocket is closely related to the performance of CcDnaN during replication and the DnaN‐mediated regulation process. Moreover, assessments of clamp competition showed that DNA does not compete with protein partners when binding to the novel pocket. Overall, our structural and biochemical analyses provide strong evidence that CcDnaN employs a noncanonical protein association pattern. [ABSTRACT FROM AUTHOR]

Abstract Phosphopantothenoylcysteine (PPC) synthetase (PPCS) catalyzes nucleoside triphosphate-dependent condensation reaction between 4′-phosphopantothenate (PPA) and l -cysteine to form PPC in CoA biosynthesis. The catalytic mechanism of PPCS has not been resolved yet. Coenzyme A biosynthesis protein 2 (Cab2) possesses activity of PPCS in Saccharomyces cerevisiae. Our enzymatic assays suggest that Cab2 could utilize both ATP and CTP to activate PPA in vitro. The results of isothermal titration calorimetry indicate that PPA, CTP, and ATP could bind to Cab2 individually, with PPA having the highest binding affinity. To provide further insight into the catalytic mechanism of Cab2, we determined the crystal structures of Cab2 and its complex with PPA, the reaction intermediate 4′-phosphopantothenoyl-CMP, the final reaction product PPC, and the product analogue phosphopantothenoylcystine. Except for PPA, all other ligands were generated in situ and present in the active-site pocket of Cab2. Structures of Cab2 in complex with ligands provide insight into substrates binding and its catalytic mechanism. Analysis of structures indicates that the carboxyl of PPA-moiety of ligands and the γ-amino group of Asn97 possess different conformations in these complex structures. The cysteine/cystine/serine selectivity assays for Cab2 indicate that the amino group rather than the thiol group of l -cysteine attacks the carbonyl of 4′-phosphopantothenoyl-CMP to form PPC. Based on structural and biochemical data, the catalytic mechanism of Cab2 was proposed for the first time. Graphical Abstract Unlabelled Image Highlights • PPCS catalyzes the formation of PPC from PPA and l -cysteine in CoA biosynthesis. • The catalytic mechanism of PPCS remains unclear. • Cab2 could utilize both ATP and CTP to activate PPA • Both PPA and CTP could bind to Cab2 separately. • The ligands and the residues possess different conformations in complex structures. [ABSTRACT FROM AUTHOR]

Abstract By bearing a papain-like core structure and a cysteine-based catalytic triad, deamidase can convert glutamine to glutamic acid or asparagine to aspartic acid to modify the functions of host target proteins resulting in the blocking of eukaryotic host cell function. Legionella pneumophila effector Lpg2148 (MvcA) is a deamidase, a structural homolog of cycle inhibiting factor (Cif) effectors. Lpg2148 and Cif effectors are functionally diverse, with Lpg2148 only catalyzing ubiquitin but not NEDD8. However, a detailed understanding of substrate specificity is still missing. Here, we resolved the crystal structure of Lpg2148 at 2.5 Å resolution and obtained rigid-body modeling of Lpg2148 with C-terminus deleted ubiquitin (1–68) (ubΔc) complex using HADDOCK, which shows that the C-terminus of ubiquitin is flexible in recognition. We also conducted the truncated analysis to demonstrate that Leu71 of ubiquitin is necessary for its interaction with Lpg2148. Moreover, Val33 of Lpg2148 at the edge of a channel plays a vital role in the interaction and is limited by the length of the C-terminus of ubiquitin, which may help to explain the selectivity of ubiquitin over NEDD8. In summary, these results enrich our knowledge of substrate recognition of deamidase. Graphical abstract Image 1 Highlights • Docking of Lpg2148/ub∆c model provides a clue that the C-terminus of ubiquitin plays a vital role in interaction. • The Leu71 in C-terminus of ubiquitin is necessary for interaction and His68 is important in deamidation. • The Val33 of Lpg2148 plays a vital role in the interaction and is limited by the length of the C-terminus of ubiquitin. [ABSTRACT FROM AUTHOR]

Thiolases are vital enzymes which participate in both degradative and biosynthetic pathways. Biosynthetic thiolases catalyze carbon–carbon bond formation by a Claisen condensation reaction. The cytoplasmic acetoacetyl‐CoA thiolase from Saccharomyces cerevisiae, ERG10, catalyses carbon–carbon bond formation in the mevalonate pathway. The structure of a S. cerevisiae biosynthetic thiolase has not previously been reported. Here, crystal structures of apo ERG10 and its Cys91Ala variant were solved at resolutions of 2.2 and 1.95 Å, respectively. The structure determined shows that ERG10 shares the characteristic thiolase superfamily fold, with a similar active‐site architecture to those of type II thiolases and a similar binding pocket, apart from Ala159 at the entrance to the pantetheine‐binding cavity, which appears to be a determinant of the poor binding ability of the substrate. Moreover, comparative binding‐pocket analysis of molecule B in the asymmetric unit of the apo structure with that of the CoA‐bound complex of human mitochondrial acetoacetyl‐CoA thiolase indicates the canonical binding mode of CoA. Furthermore, the steric hindrance revealed in a structural comparison of molecule A with the CoA‐bound form raise the possibility of conformational changes that are associated with substrate binding. [ABSTRACT FROM AUTHOR]

The versatility of Hsp90 can be attributed to the variety of co-chaperone proteins that modulate the role of Hsp90 in many cellular processes. As a co-chaperone of Hsp90, Cpr7 is essential for accelerating the cell growth in an Hsp90-containing trimeric complex. Here, we report the crystal structure of Cpr7 at a resolution of 1.8 Å. It consists of an N-terminal PPI domain and a C-terminal TPR domain, and exhibits a U-shape conformation. Our studies revealed the aggregation state of Cpr7 in solution and the interaction properties between Cpr7 and the MEEVD sequence from the C-terminus of Hsp90. In addition, the structure and sequence analysis between Cpr7 and homologues revealed the structure basis both for the function differences between Cpr6 and Cpr7 and the functional complements between Cns1 and Cpr7. Our studies facilitate the understanding of Cpr7 and provide decent insights into the molecular mechanisms of the Hsp90 co-chaperone pathway. [ABSTRACT FROM AUTHOR]

Synaptotagmins constitute a family of multifunctional integral membrane proteins found predominantly on vesicles in neural and endocrine tissues. 17 isoforms of synaptotagmin family in mammals have been identified, 7 isoforms among them are known to be able to bind Ca 2+ via their C2 domains. This study presents the crystal structure of the first C2 domain (C2A domain) of synaptotagmin 5 complexed with Ca 2+ at 1.90 Å resolution. Comparison of the Ca 2+ -binding pocket of synaptotagmin 5 C2A domain with other synaptotagmin C2 domains demonstrated that a serine residue locating at Ca 2+ -binding loop probably responsible to the conformational variation of Ca 2+ -binding pocket, and thus impacts the Ca 2+ -binding mechanism of C2 domain, which is verified by structural analysis of the serine mutant and Ca 2+ -binding assays via isothermal titration calorimetry. Alteration of Ca 2+ -binding mechanism might be correlated with different Ca 2+ response rates of synaptotagmins, which is the basis of the functions of synaptotagmins in regulating various types of Ca 2+ -triggered vesicle-membrane fusion processes. [ABSTRACT FROM AUTHOR]

It has been reported that tumor growth and proliferation correspond to mitochondrial dysfunction and that the tumor cellular microenvironment plays a key role in tumor progression, representing an area that might be manipulated to confer therapeutic anti-tumor benefits. In this article, we have identified mitochondrial genes, largely nuclear-encoded genes, which are differentially expressed in breast cancer epithelial and stromal cells compared to cells from normal breast tissues. We determined that gene expression of the mitochondrial membrane respiratory chain complex I and IV and ATP synthesis were reduced in both in epithelial and stromal cancer cells compared to normal breast cells. We also found transport-related genes were significantly more highly expressed in breast cancer epithelial cells. Our data also suggest that mitochondria are likely to proliferate in breast cancer stromal cells, which is supported by the observation that MRPL12, POLG, and RNASEH1 are all up-regulated in cancerous stromal cells. In addition, we present an improved simulated annealing algorithm, SANetWalker, which can be used to detect the functional module. At the same time, this method has a minimal effect on network topology and can be used to identify the highest confidence functional module. Using SANetWalker, we obtained the highest confidence (90%) functional module with a fumarate hydratase (FH)-centered network with 40 nodes and 107 edges. Functional analysis revealed that glutamine metabolism genes were significantly up-regulated in both epithelial and stromal cells from breast cancer tissues, which implicates glutamine metabolism in breast cancer growth and metastasis. [ABSTRACT FROM AUTHOR]

Summary Histone chaperones are critical for guiding specific post-transcriptional modifications of histones, safeguarding the histone deposition (or disassociation) of nucleosome (dis)assembly, and regulating chromatin structures to change gene activities. HAT1-interacting factor 1 (Hif1) has been reported to be an H3-H4 chaperone and to be involved in telomeric silencing and nucleosome (dis)assembly. However, the structural basis for the interaction of Hif1 with histones remains unknown. Here, we report the complex structure of Hif1 binding to H2A-H2B for uncovering the chaperone specificities of Hif1 on binding to both the H2A-H2B dimer and the H3-H4 tetramer. Our findings reveal that Hif1 interacts with the H2A-H2B dimer and the H3-H4 tetramer via distinct mechanisms, suggesting that Hif1 is a pivotal scaffold on alternate binding of H2A-H2B and H3-H4. These specificities are conserved features of the Sim3-Hif1-NASP interrupted tetratricopeptide repeat proteins, which provide clues for investigating their potential roles in nucleosome (dis)assembly. [ABSTRACT FROM AUTHOR]

l -amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l -amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris ( pv LAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pv LAAD does not resemble typical LAAOs. Instead it, is similar to d -amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pv LAAD could generate a significant amount of hydrogen peroxide in vitro , suggesting it could use dioxygen to directly re-oxidize FADH 2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications. [ABSTRACT FROM AUTHOR]

Clathrin-coated vesicles (CCVs) play critical roles in multiple cellular processes, including nutrient uptake, endosome/lysosome biogenesis, pathogen invasion, regulation of signalling receptors, etc. Saccharomyces cerevisiae Ent5 ( Sc Ent5) is one of the two major adaptors supporting the CCV-mediated TGN/endosome traffic in yeast cells. However, the classification and phosphoinositide binding characteristic of Sc Ent5 remain elusive. Here we report the crystal structures of the Sc Ent5 N-terminal domain, and find that Sc Ent5 contains an insertion α′ helix that does not exist in other ENTH or ANTH domains. Furthermore, we investigate the classification of Sc Ent5-N 31−191 by evolutionary history analyses and structure comparisons, and find that the Sc Ent5 N-terminal domain shows different phosphoinositide binding property from r Epsin1 and r CALM. Above results facilitate the understanding of the Sc Ent5-mediated vesicle coat formation process. [ABSTRACT FROM AUTHOR]

The paper by Liu et al. is corrected. [ABSTRACT FROM AUTHOR]

Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription. In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length Mj Spt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region was found on the Spt4:Spt5 surface in both crystal forms, and identified as double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, the Spt4:Spt5-DNA binding model was built for the first time. [ABSTRACT FROM AUTHOR]

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors in Staphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeRDBD) was solved at 2.5 Å resolution. The structure reveals that SaeRDBD exists as a monomer and has the canonical winged helix-turn-helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix-turn-helix module were verified to be important for binding to the P1 promoter in vitro and for the physiological function of SaeR in vivo. [ABSTRACT FROM AUTHOR]

After deadenylation and decapping, cytoplasmic mRNA can be digested in two opposite directions: in the 5′-3′ direction by Xrn1 or in the 3′-5′ direction by the exosome complex. Recently, a novel 3′-5′ RNA-decay pathway involving Dis3l2 has been described that differs from degradation by Xrn1 and the exosome. The product of the Schizosaccharomyces pombe gene SPAC2C4.07c was identified as a homologue of human Dis3l2. In this work, the 2.8 Å resolution X-ray crystal structure of S. pombe Dis3l2 ( SpDis3l2) is reported, the conformation of which is obviously different from that in the homologous mouse Dis3l2-RNA complex. Fluorescence polarization assay experiments showed that RNB and S1 are the primary RNA-binding domains and that the CSDs (CSD1 and CSD2) play an indispensable role in the RNA-binding process of SpDis3l2. Taking the structure comparison and mutagenic experiments together, it can be inferred that the RNA-recognition pattern of SpDis3l2 resembles that of its mouse homologue rather than that of the Escherichia coli RNase II-RNA complex. Furthermore, a drastic conformation change could occur following the binding of the RNA substrate to SpDis3l2. [ABSTRACT FROM AUTHOR]

Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants. [ABSTRACT FROM AUTHOR]

Phospholipase A2 (PLA2) is an important component in snake venoms. Here, an acidic PLA2, designated PA2‐Vb was isolated from the Trimeresurus stejnegeri snake venom. PA2‐Vb acts on a protease‐activated receptor (PAR‐1) to evoke Ca2+ release through the inositol 1,4,5‐trisphosphate receptor (IP3R) and induces mouse aorta contraction. PAR‐1, phospholipase C and IP3R inhibitors suppressed PA2‐Vb‐induced aorta contraction. The crystal structure reveals that PA2‐Vb has the typical fold of most snake venom PLA2. Several PEG molecules bond to a positively charged pocket. The finding offers a novel pharmacological basis of the structure for investigating the PAR‐1 receptor and suggests potential applications for PA2‐Vb in the vascular system. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC: 1.2.1.12) is an essential enzyme in the glycolytic pathway. However, recent evidence demonstrates that GAPDH displays a range of new functions unrelated to its glycolytic function. GAPDH has long been known as a 3′ AU-rich element-binding protein; however, its RNA recognition mechanism is still not well understood. Here, we present the first crystal structure of GAPDH3 from Saccharomyces cerevisiae and identify its RNA-binding specificity and propose an RNA recognition model based on structural and biochemical studies. This study sheds light on the RNA-binding mechanism of GAPDH3 and contributes to a better understanding of the molecular mechanisms of its RNA-related functions. © 2014 IUBMB Life, 66(9):631-638, 2014 [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a highly versatile pathogen that can infect human tissue by producing a large arsenal of virulence factors that are tightly regulated by a complex regulatory network. Rot, which shares sequence similarity with SarA homologues, is a global regulator that regulates numerous virulence genes. However, the recognition model of Rot for the promoter region of target genes and the putative regulation mechanism remain elusive. In this study, the 1.77 Å resolution X-ray crystal structure of Rot is reported. The structure reveals that two Rot molecules form a compact homodimer, each of which contains a typical helix-turn-helix module and a β-hairpin motif connected by a flexible loop. Fluorescence polarization results indicate that Rot preferentially recognizes AT-rich dsDNA with ∼30-base-pair nucleotides and that the conserved positively charged residues on the winged-helix motif are vital for binding to the AT-rich dsDNA. It is proposed that the DNA-recognition model of Rot may be similar to that of SarA, SarR and SarS, in which the helix-turn-helix motifs of each monomer interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves. Interestingly, the structure shows that Rot adopts a novel dimerization model that differs from that of other SarA homologues. As expected, perturbation of the dimer interface abolishes the dsDNA-binding ability of Rot, suggesting that Rot functions as a dimer. In addition, the results have been further confirmed in vivo by measuring the transcriptional regulation of α-toxin, a major virulence factor produced by most S. aureus strains. [ABSTRACT FROM AUTHOR]

Summary: Trypanosoma brucei protein arginine methyltransferase 7 (TbPRMT7) exclusively generates monomethylarginine (MMA), which directs biological consequences distinct from that of symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA). However, determinants controlling the strict monomethylation activity are unknown. We present the crystal structure of the TbPRMT7 active core in complex with S-adenosyl-L-homocysteine (AdoHcy) and a histone H4 peptide substrate. In the active site, residues E172, E181, and Q329 hydrogen bond the guanidino group of the target arginine and align the terminal guanidino nitrogen in a position suitable for nucleophilic attack on the methyl group of S-adenosyl-L-methionine (AdoMet). Structural comparisons and isothermal titration calorimetry data suggest that the TbPRMT7 active site is narrower than those of protein arginine dimethyltransferases, making it unsuitable to bind MMA in a manner that would support a second turnover, thus abolishing the production of SDMA and ADMA. Our results present the structural interpretations for the monomethylation activity of TbPRMT7. [ABSTRACT FROM AUTHOR]

Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6) is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH). The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6. [ABSTRACT FROM AUTHOR]

RraB, an inhibitor of the essential endoribonuclease RNE in Escherichia coli, is essential in regulating the abundance of RNA by directly interacting with RNE. In this study, RraB from E. coli was cloned, expressed, purified and crystallized. The crystals belonged to space group P212121, with unit-cell parameters a = 58.59, b = 58.34, c = 156.95 Å. X-ray diffraction data were collected to a resolution of 2.9 Å. Analysis of the native Patterson map revealed a peak of ∼37% the height of the origin peak in the ν = 0.5 Harker section, suggesting twofold noncrystallographic symmetry parallel to the b crystallographic axis. The Matthews coefficient and the solvent content were estimated to be 4.09 Å3 Da−1 and 69.94%, respectively, assuming the presence of two molecules in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Highlights: [•] MHF forms a hetero-octamer with each tetramer binding to one molecule of FANCM-F. [•] The missing C-ends of MHF1 in crystal structures were successfully restored. [•] The positively charged patch is joined up by the octameric structure of MHF. [•] The negatively charged area on the patch is concealed by the C-terminus of MHF1. [•] Two FA core complexes work together to finish their functional assembly. [ABSTRACT FROM AUTHOR]

The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1. [ABSTRACT FROM AUTHOR]

AhV_aPA, the acidic PLA2 purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K+-induced contraction with an EC50 in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca2+ releasing from Ca2+ store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca2+ release from SR is related to the activation of inositol trisphosphate receptors (IP3Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with heparin, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA2 and provide a novel pathogenesis of A. halys pallas venom. [ABSTRACT FROM AUTHOR]

Phosphoribosyl pyrophosphate synthetase isoform 1 (PRS1) has an essential role in the de novo and salvage synthesis of human purine and pyrimidine nucleotides. The dysfunction of PRS1 will dramatically influence nucleotides' concentration in patient's body and lead to different kinds of disorders (such as hyperuricemia, gout and deafness). The D52H missense mutation of PRS1 will lead to a conspicuous phosphoribosyl pyrophosphate content elevation in the erythrocyte of patients and finally induce hyperuricemia and serious gout. In this study, the enzyme activity analysis indicated that D52H-mutant possessed similar catalytic activity to the wild-type PRS1, and the 2.27 Å resolution D52H-mutant crystal structure revealed that the stable interaction network surrounding the 52 position of PRS1 would be completely destroyed by the substitution of histidine. These interaction variations would further influence the conformation of ADP-binding pocket of D52H-mutant and reduced the inhibitor sensitivity of PRS1 in patient's body. © 2013 IUBMB Life, 65(6):518-525, 2013. [ABSTRACT FROM AUTHOR]

A snake venom thrombin-like enzyme (SVTLE) from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the Aα and Bβ chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533 Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of 1.75 Å. A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the 99-loop, and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC of 147 nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca] measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca releasing from Ca store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom. [ABSTRACT FROM AUTHOR]

Abstract: Ap4A hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17), an enzyme involved in a number of biological processes, is characterized as cleaving the polyphosphate chain at the fourth phosphate from the bound adenosine moiety. This paper presents the crystal structure of wild-type and E58A mutant human Ap4A hydrolase. Similar to the canonical Nudix fold, human Ap4A hydrolase shows the common αβα-sandwich architecture. Interestingly, two sulfate ions and one diphosphate coordinated with some conserved residues were observed in the active cleft, which affords a better understanding of a possible mode of substrate binding. [Copyright &y& Elsevier]

Human RNA-binding protein (HuR), a ubiquitously expressed member of the Hu protein family, plays an important role in mRNA degradation and has been implicated as a key post-transcriptional regulator. HuR contains three RNA-recognition motif (RRM) domains. The two N-terminal tandem RRM domains can selectively bind AU-rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly-A tail of target mRNA and other ligands. Here, the X-ray structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form was solved at 2.9 Å resolution. The crystal structure of RRM1/2 complexed with target mRNA was also solved at 2.0 Å resolution; comparisons of the two structures show that HuR RRM1/2 undergoes conformational changes upon RNA binding. Fluorescence polarization assays (FPA) were used to study the protein-RNA interactions. Both the structure and the FPA analysis indicated that RRM1 is the primary ARE-binding domain in HuR and that the conformational changes induce subsequent contacts of the RNA substrate with the inter-domain linker and RRM2 which greatly improve the RNA-binding affinity of HuR. [ABSTRACT FROM AUTHOR]

Phosphorylated derivatives of phosphatidylinositol (PtdIns), also called phosphoinositides (PIPs), are basic components of membrane-associated signalling systems. A family of PtdIns-transfer proteins (PITPs) called the Sec14 family have been predicted to form a set of functional modules that can sense different types of lipid metabolism and transmit the information to the PIP signalling system. In eukaryotic cells, the Sec14 family exhibits a wide diversity of activity, but the structural basis of this diversity remains unclear. In the present study, the dimeric structure of Sfh3 (Sec14 family homologue 3 in yeast) is reported for the first time and differs from the Sec14 proteins reported to date, all of which are monomeric. Some variations in the binding pocket of Sfh3 were observed and the dimer interface was identified and proposed to provide a link between dimer-monomer state changes and PtdIns binding. Together, these structural changes and the oligomeric state transformation of Sfh3 support ideas of diversity within the Sec14 family and provide some new clues to function. [ABSTRACT FROM AUTHOR]

3-Oxoacyl-(acyl-carrier-protein) reductase (OAR1 or FabG, EC.1.1.1.100) is responsible for the first reductive step in fatty acid biosynthesis using Nicotinamide Adenine Dinucleotide Phosphate (NADPH) as a cofactor. Recent studies suggest there is a fatty acid synthetase II pathway that consists of a series of separate enzymes in yeast mitochondrion. Here, we present the crystal structure of the yeast mitochondria OAR1 (ymtOAR1) alone in apo-form at 2.60 Å and complexed with NADPH at 2.10 Å resolution. Unlike the reported tetrameric OARs, ymtOAR1 forms a homodimer due to the different fold. The enzyme generates conformational changes upon NADPH binding to the active site. Moreover, two different cofactor-binding patterns are observed from two forms of complex crystals, and structural analysis implies the adenine end of cofactor may recognize enzyme prior to nicotinaminde end. Additionally, biochemical studies suggest Arg14 is important for cofactor recognition of ymtOAR1. © 2013 IUBMB Life, 65(2)154-162, 2013. [ABSTRACT FROM AUTHOR]

Abstract: Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of d-mannonate to 2-keto-3-deoxygluconate. It is the third enzyme in the pathway for dissimilation of d-glucuronate to 2-keto-3-deoxygluconate involving in the Entner–Doudoroff pathway in certain bacterial and archaeal species. ManD from Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by sequence analysis. To evaluate the impact of this insert sequence on the catalytic efficiency, we solved the crystal structures of ManD from Escherichia coli strain K12 and its complex with d-mannonate, which reveal that this insert sequence forms two α helices locating above the active site. The two insert α helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site. Site-directed mutations and enzymatic activity assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common inducer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays. [Copyright &y& Elsevier]

The yeast Paf1 complex (Paf1C), which is composed of the proteins Paf1, Cdc73, Ctr9, Leo1 and Rtf1, accompanies RNA polymerase II from the promoter to the 3′-end formation site of mRNA- and snoRNA-encoding genes. As one of the first identified subunits of Paf1C, yeast Cdc73 (yCdc73) takes part in many transcription-related processes, including binding to RNA polymerase II, recruitment and activation of histone-modification factors and communication with other transcriptional activators. The human homologue of yCdc73, parafibromin, has been identified as a tumour suppressor linked to breast, renal and gastric cancers. However, the functional mechanism of yCdc73 has until recently been unclear. Here, a 2.2 Å resolution crystal structure of the highly conserved C-terminal region of yCdc73 is reported. It revealed that yCdc73 appears to have a GTPase-like fold. However, no GTPase activity was observed. The crystal structure of yCdc73 will shed new light on the modes of function of Cdc73 and Paf1C. [ABSTRACT FROM AUTHOR]

Abstract: Human Pax2 transactivation domain-interacting protein (hPTIP), containing six BRCT domains, is an essential protein required for the IR induced DDR process with an unclear role. Here we report that the tandem BRCT5–BRCT6 domain of hPTIP recognizes the γH2AX tail, and this interaction depends on the phosphorylation of H2AX Ser139 and binding with the carboxyl ending peptide to the aminoacyl ending peptide. The 2.15Å crystal structure of hPTIP BRCT5/6–γH2AX complex and mutation analysis provide molecular evidence for direct interactions between PTIP and γH2AX. This interaction proffers a new clue to identify the role of PTIP in DDR pathways. Structured summary of protein interactions: PTIP and gamma H2AX bind by fluorescence polarization spectroscopy (View Interaction: 1, 2, 3, 4, 5, 6). PTIP and gamma H2AX bind by X-ray crystallography (View interaction). [Copyright &y& Elsevier]

p185her2/neu belongs to the ErbB receptor tyrosine kinase family, which has been associated with human breast, ovarian, and lung cancers. Targeted therapies employing ectodomain-specific p185her2/neu monoclonal antibodies (mAbs) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185her2/neu mAbs are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185her2/neu-overexpressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185her2/neu, which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185her2/neu. We propose a structure-based model in which chA21 cross-links two p185her2/neu molecules on separate homo- or heterodimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAbs to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a "physiologic process." [ABSTRACT FROM AUTHOR]