Insights from the Structure of an Active Form of Bacillus thuringiensis Cry5B.

The crystal protein Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis, has been demonstrated to have excellent anthelmintic activity. While a previous structure of the three-domain core region of Cry5B(112-698) had been reported, this structure lacked a key N-terminal extension critical to function. Here we report the structure of Cry5B(27-698) containing this N-terminal extension. This new structure adopts a distinct quaternary structure compared to the previous Cry5B(112-698) structure, and also exhibits a change in the conformation of residues 112-140 involved in linking the N-terminal extension to the three-domain core by forming a random coil and an extended α-helix. A role for the N-terminal extension is suggested based on a computational model of the tetramer with the conformation of residues 112-140 in its alternate α-helix conformation. Finally, based on the Cry5B(27-698) structure, site-directed mutagenesis studies were performed on Tyr495, which revealed that having an aromatic group or bulky group at this residue 495 is important for Cry5B toxicity.

Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.

Expression, purification, crystallization and preliminary X-ray analysis of full-length human RIG-I.

The human innate immune system can detect invasion by microbial pathogens through pattern-recognition receptors that recognize structurally conserved pathogen-associated molecular patterns. Retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) are one of the two major families of pattern-recognition receptors that can detect viral RNA. RIG-I, belonging to the RLH family, is capable of recognizing intracellular viral RNA from RNA viruses, including influenza virus and Ebola virus. Here, full-length human RIG-I (hRIG-I) was cloned in Escherichia coli and expressed in a recombinant form with a His-SUMO tag. The protein was purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.85 Å resolution; the crystal belonged to space group F23, with unit-cell parameters a = b = c = 216.43 Å, α = ß = γ = 90°.

Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer.

DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson-Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.

Structural basis of FliG-FliM interaction in Helicobacter pylori.

FliG and FliM are switch proteins that regulate the rotation and switching of the flagellar motor. Several assembly models for FliG and FliM have recently been proposed; however, it remains unclear whether the assembly of the switch proteins is conserved among different bacterial species. We applied a combination of pull-down, thermodynamic and structural analyses to characterize the FliM-FliG association from the mesophilic bacterium Helicobacter pylori. FliM binds to FliG with micromolar binding affinity, and their interaction is mediated through the middle domain of FliG (FliGM ), which contains the EHPQR motif. Crystal structures of the middle domain of H. pylori FliM (FliM(M)) and FliG(M) -FliM(M) complex revealed that FliG binding triggered a conformational change of the FliM α3-α1' loop, especially Asp130 and Arg144. We furthermore showed that various highly conserved residues in this region are required for FliM-FliG complex formation. Although the FliM-FliG complex structure displayed a conserved binding mode when compared with Thermotoga maritima, variable residues were identified that may contribute to differential binding affinities across bacterial species. Comparison of the thermodynamic parameters of FliG-FliM interactions between H. pylori and Escherichia coli suggests that molecular basis and binding properties of FliM to FliG is likely different between these two species.

Structure of Crimean-Congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage.

Crimean-Congo hemorrhagic fever, a severe hemorrhagic disease found throughout Africa, Europe, and Asia, is caused by the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Nairovirus genus of the Bunyaviridae family. Its genome of three single-stranded RNA segments is encapsidated by the nucleocapsid protein (CCHFV N) to form the ribonucleoprotein complex. This ribonucleoprotein complex is required during replication and transcription of the viral genomic RNA. Here, we present the crystal structures of the CCHFV N in two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit with the base of the globular body of the adjacent subunit stabilizes the helical organization of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. By incubation with primer-length ssRNAs, we also obtained the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change of CCHFV N upon deoligomerization results in the exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. Thus, cleavage of CCHFV N by host cell caspase-3 appears to be crucial for controlling viral RNA synthesis and represents an important host defense mechanism against CCHFV infection.

Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling.

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.

Expression, purification, crystallization and preliminary X-ray analysis of Plasmodium falciparum GTP:AMP phosphotransferase.

Adenylate kinases (AKs) are phosphotransferase enzymes that catalyze the interconversion of adenine nucleotides, thereby playing an important role in energy metabolism. In Plasmodium falciparum, three AK isoforms, namely PfAK1, PfAK2 and GTP:AMP phosphotransferase (PfGAK), have been identified. While PfAK1 and PfAK2 catalyse the conversion of ATP and AMP to two molecules of ADP, PfGAK exhibits a substrate preference for GTP and AMP and does not accept ATP as a substrate. PfGAK was cloned and expressed in Escherichia coli and purified using two-step chromatography. Brown hexagonal crystals of PfGAK were obtained and a preliminary diffraction analysis was performed. X-ray diffraction data for a single PfGAK crystal were processed to 2.9 Å resolution in space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 123.49, c = 180.82 Å, α = ß = 90, γ = 120°.

The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus.

RNA helicases of the DEAD (Asp-Glu-Ala-Asp)-box family of proteins are involved in many aspects of RNA metabolism from transcription to RNA decay, but most of them have also been shown to be multifunctional. The DEAD-box helicase DDX5 of host cells has been shown to interact with the RNA-dependent RNA polymerase (NS5B) of HCV (hepatitis C virus). In the present study, we report the presence of two independent NS5B-binding sites in DDX5, one located at the N-terminus and another at the C-terminus. The N-terminal fragment of DDX5, which consists of the first 305 amino acids and shall be referred as DDX5-N, was expressed and crystallized. The crystal structure shows that domain 1 (residues 79-303) of DDX5 contains the typical features found in the structures of other DEAD-box helicases. DDX5-N also contains the highly variable NTR (N-terminal region) of unknown function and the crystal structure reveals structural elements in part of the NTR, namely residues 52-78. This region forms an extensive loop and an α-helix. From co-immunoprecipitation experiments, the NTR of DDX5-N was observed to auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-51 residues in NTR and the NS5B-binding site in DDX5-N. Furthermore, NMR investigations reveal that there is a direct interaction between DDX5 and NS5B in vitro.

Structural studies of intermediates along the cyclization pathway of Aplysia ADP-ribosyl cyclase.

Cyclic ADP-ribose (cADPR) is a calcium messenger that can mobilize intracellular Ca²âº stores and activate Ca²âº influx to regulate a wide range of physiological processes. Aplysia cyclase is the first member of the ADP-ribosyl cyclases identified to catalyze the cyclization of NAD⁺ into cADPR. The catalysis involves a two-step reaction, the elimination of the nicotinamide ring and the cyclization of the intermediate resulting in the covalent attachment of the purine ring to the terminal ribose. Aplysia cyclase exhibits a high degree of leniency towards the purine base of its substrate, and the cyclization reaction takes place at either the N1- or the N7-position of the purine ring. To decipher the mechanism of cyclization in Aplysia cyclase, we used a crystallization setup with multiple Aplysia cyclase molecules present in the asymmetric unit. With the use of natural substrates and analogs, not only were we able to capture multiple snapshots during enzyme catalysis resulting in either N1 or N7 linkage of the purine ring to the terminal ribose, we were also able to observe, for the first time, the cyclized products of both N1 and N7 cyclization bound in the active site of Aplysia cyclase.

Induced-fit upon ligand binding revealed by crystal structures of the hot-dog fold thioesterase in dynemicin biosynthesis.

Dynemicins are structurally related 10-membered enediyne natural products isolated from Micromonospora chernisa with potent antitumor and antibiotic activity. The early biosynthetic steps of the enediyne moiety of dynemicins are catalyzed by an iterative polyketide synthase (DynE8) and a thioesterase (DynE7). Recent studies indicate that the function of DynE7 is to off-load the linear biosynthetic intermediate assembled on DynE8. Here, we report crystal structures of DynE7 in its free form at 2.7 Å resolution and of DynE7 in complex with the DynE8-produced all-trans pentadecen-2-one at 2.1 Å resolution. These crystal structures reveal that upon ligand binding, significant conformational changes throughout the substrate-binding tunnel result in an expanded tunnel that traverses an entire monomer of the tetrameric DynE7 protein. The enlarged inner segment of the channel binds the carbonyl-conjugated polyene mainly through hydrophobic interactions, whereas the putative catalytic residues are located in the outer segment of the channel. The crystallographic information reinforces an unusual catalytic mechanism that involves a strictly conserved arginine residue for this subfamily of hot-dog fold thioesterases, distinct from the typical mechanism for hot-dog fold thioesterases that utilizes an acidic residue for catalysis.

Molecular interaction of flagellar export chaperone FliS and cochaperone HP1076 in Helicobacter pylori.

Flagellar export chaperone FliS prevents premature polymerization of flagellins and is critical for flagellar assembly and bacterial colonization. Previously, a yeast 2-hybrid study identified various FliS-associated proteins in Helicobacter pylori, but the implications of these interactions are not known. Here we demonstrate the biophysical interaction of FliS (HP0753) and the uncharacterized protein HP1076 from H. pylori. HP1076 possesses a cochaperone activity that promotes the folding and chaperone activity of FliS. We further determined the crystal structures of FliS, HP1076, and the binary complex at 2.7, 1.8, and 2.7 Å resolution, respectively. HP1076 adopts a helix-rich bundle structure and interestingly shares a similar fold with a flagellin homologue, hook-associated protein, and FliS. The FliS-HP1076 complex revealed an extensive electrostatic and hydrophobic binding interface, which is distinct from the flagellin binding pocket in FliS. The helical stacking interaction between HP1076 and FliS suggests that HP1076 stabilizes 2 α helices of FliS and therefore the overall structure of the bundle. Our findings provide new insights into flagellar export chaperones and may have implications for other secretion chaperones in the type III secretion system.

Flexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications.

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180-618) by an 11-amino acid linker (169-179). The structure at 3.15 A of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B(18)NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by approximately 161 degrees with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn(2+) refined to a resolution of 2.2 A. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu(173) and Pro(174) or replacing Pro(174) with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro(176) to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.

Cutting edge: granulocyte-macrophage colony-stimulating factor is the major CD8+ T cell-derived licensing factor for dendritic cell activation.

During priming, CD8(+) T lymphocytes can induce robust maturation of dendritic cells (DCs) in a CD40-independent manner by secreting licensing factor(s). In this study, we isolate this so-far elusive licensing factor and identify it, surprisingly, as GM-CSF. This provides a new face for an old factor with a well-known supporting role in DC development and recruitment. Signaling through the GM-CSFR in ex vivo-purified DCs upregulated the expression of costimulatory molecules more efficiently than did any tested TLR agonist and provided a positive feedback loop in the stimulation of CD8(+) T cell proliferation. Combined with a variety of microbial stimuli, GM-CSF supports the formation of potent "effector" DCs capable of secreting a variety of proinflammatory cytokines that guide the differentiation of T cells during the immune response.

Expression, purification and preliminary crystallographic analysis of Pseudomonas aeruginosa RocR protein.

Pseudomonas aeruginosa RocR, an EAL-domain protein which regulates the expression of virulence genes and biofilm formation, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of RocR are reported. The X-ray diffraction data were processed to a resolution of 2.50 A. The crystals belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = 118.8, b = 118.8, c = 495.1 A, alpha = beta = 90, gamma = 120 degrees .

Mechanism of cyclizing NAD to cyclic ADP-ribose by ADP-ribosyl cyclase and CD38.

Mammalian CD38 and its Aplysia homolog, ADP-ribosyl cyclase (cyclase), are two prominent enzymes that catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger molecule responsible for regulating a wide range of cellular functions. Although both use NAD as a substrate, the cyclase produces cADPR, whereas CD38 produces mainly ADP-ribose (ADPR). To elucidate the catalytic differences and the mechanism of cyclizing NAD, the crystal structure of a stable complex of the cyclase with an NAD analog, ribosyl-2'F-2'deoxynicotinamide adenine dinucleotide (ribo-2'-F-NAD), was determined. The results show that the analog was a substrate of the cyclase and that during the reaction, the nicotinamide group was released and a stable intermediate was formed. The terminal ribosyl unit at one end of the intermediate formed a close linkage with the catalytic residue (Glu-179), whereas the adenine ring at the other end stacked closely with Phe-174, suggesting that the latter residue is likely to be responsible for folding the linear substrate so that the two ends can be cyclized. Mutating Phe-174 indeed reduced cADPR production but enhanced ADPR production, converting the cyclase to be more CD38-like. Changing the equivalent residue in CD38, Thr-221 to Phe, correspondingly enhanced cADPR production, and the double mutation, Thr-221 to Phe and Glu-146 to Ala, effectively converted CD38 to a cyclase. This study provides the first detailed evidence of the cyclization process and demonstrates the feasibility of engineering the reactivity of the enzymes by mutation, setting the stage for the development of tools to manipulate cADPR metabolism in vivo.

The functional role of a conserved loop in EAL domain-based cyclic di-GMP-specific phosphodiesterase.

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (beta/alpha)(8) barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K(m)) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K(m) correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg(2+) ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.

Structure and catalytic mechanism of the thioesterase CalE7 in enediyne biosynthesis.

The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-A crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg(37) plays a crucial role in the hydrolysis of the thioester bond, and that Tyr(29) and a hydrogen-bonded water network assist the decarboxylation of the beta-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4=C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.

Structural analysis of the recognition of the negative regulator NmrA and DNA by the zinc finger from the GATA-type transcription factor AreA.

Amongst the most common protein motifs in eukaryotes are zinc fingers (ZFs), which, although largely known as DNA binding modules, also can have additional important regulatory roles in forming protein:protein interactions. AreA is a transcriptional activator central to nitrogen metabolism in Aspergillus nidulans. AreA contains a GATA-type ZF that has a competing dual recognition function, binding either DNA or the negative regulator NmrA. We report the crystal structures of three AreA ZF-NmrA complexes including two with bound NAD(+) or NADP(+). The molecular recognition of AreA ZF-NmrA involves binding of the ZF to NmrA via hydrophobic and hydrogen bonding interactions through helices alpha1, alpha6 and alpha11. Comparison with an earlier NMR solution structure of AreA ZF-DNA complex by overlap of the AreA ZFs shows that parts of helices alpha6 and alpha11 of NmrA are positioned close to the GATA motif of the DNA, mimicking the major groove of DNA. The extensive overlap of DNA with NmrA explains their mutually exclusive binding to the AreA ZF. The presence of bound NAD(+)/NADP(+) in the NmrA-AreaA ZF complex, however, causes minimal structural changes. Thus, any regulatory effects on AreA function mediated by the binding of oxidised nicotinamide dinucleotides to NmrA in the NmrA-AreA ZF complex appear not to be modulated via protein conformational rearrangements.

Expression, purification and preliminary crystallographic analysis of recombinant human small glutamine-rich tetratricopeptide-repeat protein.

Human small glutamine-rich tetratricopeptide-repeat protein (hSGT) is a 35 kDa protein implicated in a number of biological processes that include apoptosis, cell division and intracellular cell transport. The tetratricopeptide-repeat (TPR) domain of hSGT has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of the TPR domain of hSGT is reported. X-ray diffraction data were processed to a resolution of 2.4 A. Crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 67.82, b = 81.93, c = 55.92 A, alpha = beta = gamma = 90 degrees .