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Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital-acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso-DAP and lysine. The rate-limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (k cat  = 29 s -1 and 44 s -1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC 50  = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen. DATABASES: PDB ID: 6P90., (© 2019 Federation of European Biochemical Societies.)

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin., (open access.)

Lanthanoid heteroleptic complexes with cucurbit[5]uril {Q[5]} and two dicarboxylate ligands, e.g., diglycolic acid (H 2 DGC) and glutaric acid (H 2 GT), have been investigated with six new compounds featuring a tetrametallic and dimetallic discrete structures, a one-dimensional (1D) polymer, and three two-dimensional (2D) polymers with a unique honeycomb-type topology being synthesized and structurally characterized. [La 4 (Q[5]) 3 (DGC) 2 (NO 3 ) 2 (H 2 O) 12 ][La(DGC)(H 2 O) 6 ]·7NO 3 · nH 2 O (1) has a tetrametallic structure constructed with three bis-bidentate Q[5] ligands linking two [La(DGC)(H 2 O) 2 ] + species in the middle and two [La(H 2 O) 4 (NO 3 )] 2+ species at both ends. [Ce 2 (Q[5])(DGC)(NO 3 )(H 2 O) 10 ]·3NO 3 ·4H 2 O (2) has a dimetallic structure built up with a bis-bidentate Q[5] ligand linking [Ce(DGC)(H 2 O) 3 (NO 3 )] and [Ce(H 2 O) 7 ] 3+ on each side of the Q[5] portals. [Ce 3 (Q[5]) 3 (DGC) 2 (H 2 O) 9 ][Ce(DGC)(H 2 O) 6 ] 2 ·7NO 3 · nH 2 O (3) has a 1D polymeric structure built up with bis-bidentate Q[5] ligands in-turn linking one [Ce(H 2 O) 6 ] 3+ and two [Ce(DGC)(H 2 O) 6 ] 1+ cationic species. [Ln 2 (Q[5]) 2 (GT)(H 2 O) 6 ]·4NO 3 · nH 2 O [Ln = La (4), Ce (5) and Nd (6)] have similar 2D polymeric structures built up with two types of 9-fold coordinated Ln polyhedra linked by Q[5] via bis-bidentate carbonyl groups on both sides forming 1D chains which are further connected by bridging GT 2- ligands to form 2D polymers with a unique honeycomb-type topology. Their vibrational modes and electronic structures have also been investigated.

Benzene and its derivatives form a class of priority pollutants whose exposure poses grave risk to human health. Since benzene lacks active functional groups, devising specific sensors for its direct detection from a milieu of aromatics has remained a daunting task. Here, we report three engineered protein-based biosensors that exclusively and specifically detect benzene and its derivatives up to a detection limit of 0.3 ppm. Further, the biosensor design has been engineered to create templates that possess the ability to specifically discriminate between alkyl substituted benzene derivatives; such as toluene, m-xylene, and mesitylene. Interference tests with simulated wastewater samples reveal that the engineered biosensors can selectively detect a specific benzene compound in water samples containing a milieu of high concentrations of commonly occurring pollutants. This work demonstrates the potential of structure guided protein engineering as a competent strategy toward design of selective biosensors for direct detection of benzene group of pollutants from real time environmental samples.

The different colors of light emitted by bioluminescent beetles that use an identical substrate and chemiexcitation reaction sequence to generate light remain a challenging and controversial mechanistic conundrum. The crystal structures of two beetle luciferases with red- and blue-shifted light relative to the green yellow light of the common firefly species provide direct insight into the molecular origin of the bioluminescence color. The structure of a blue-shifted green-emitting luciferase from the firefly Amydetes vivianii is monomeric with a structural fold similar to the previously reported firefly luciferases. The only known naturally red-emitting luciferase from the glow-worm Phrixothrix hirtus exists as tetramers and octamers. Structural and computational analyses reveal varying aperture between the two domains enclosing the active site. Mutagenesis analysis identified two conserved loops that contribute to the color of the emitted light. These results are expected to advance comparative computational studies into the conformational landscape of the luciferase reaction sequence., Competing Interests: The authors declare that they have no conflict of interest.

Dihydrodipicolinate synthase (DHDPS) is critical to the production of lysine through the diaminopimelate (DAP) pathway. Elucidation of the function, regulation and structure of this key class I aldolase has been the focus of considerable study in recent years, given that the dapA gene encoding DHDPS has been found to be essential to bacteria and plants. Allosteric inhibition by lysine is observed for DHDPS from plants and some bacterial species, the latter requiring a histidine or glutamate at position 56 (Escherichia coli numbering) over a basic amino acid. Structurally, two DHDPS monomers form the active site, which binds pyruvate and (S)-aspartate β-semialdehyde, with most dimers further dimerising to form a tetrameric arrangement around a solvent-filled centre cavity. The architecture and behaviour of these dimer-of-dimers is explored in detail, including biophysical studies utilising analytical ultracentrifugation, small-angle X-ray scattering and macromolecular crystallography that show bacterial DHDPS tetramers adopt a head-to-head quaternary structure, compared to the back-to-back arrangement observed for plant DHDPS enzymes. Finally, the potential role of pyruvate in providing substrate-mediated stabilisation of DHDPS is considered.

The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a diversity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the diversity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output.

Phenolic aromatic compounds are a major source of environmental pollution. Currently there are no in situ methods for specifically and selectively detecting these pollutants. Here, we exploit the nature's biosensory machinery by employing Acinetobacter calcoaceticus NCIB8250 protein, MopR, as a model system to develop biosensors for selective detection of a spectrum of these pollutants. The X-ray structure of the sensor domain of MopR was used as a scaffold for logic-based tunable biosensor design. By employing a combination of in silico structure guided approaches, mutagenesis and isothermal calorimetric studies, we were able to generate biosensor templates, that can selectively and specifically sense harmful compounds like chlorophenols, cresols, catechol, and xylenols. Furthermore, the ability of native protein to selectively sense phenol as the primary ligand was also enhanced. Overall, this methodology can be extended as a suitable framework for development of a series of exclusive biosensors for accurate and selective detection of aromatic pollutants from real time environmental samples.

A recent publication by Seng et al. in this journal reports the crystallographic structure of refolded, full-length SMN protein and two disease-relevant derivatives thereof. Here, we would like to suggest that at least two of the structures reported in that study are incorrect. We present evidence that one of the associated crystallographic datasets is derived from a crystal of the bacterial Sm-like protein Hfq and that a second dataset is derived from a crystal of the bacterial Gab protein. Both proteins are frequent contaminants of bacterially overexpressed proteins which might have been co-purified during metal affinity chromatography. A third structure presented in the Seng et al. paper cannot be examined further because neither the atomic coordinates, nor the diffraction intensities were made publicly available. The Tudor domain protein SMN has been shown to be a component of the SMN complex, which mediates the assembly of RNA-protein complexes of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). Importantly, this activity is reduced in SMA patients, raising the possibility that the aetiology of SMA is linked to RNA metabolism. Structural studies on diverse components of the SMN complex, including fragments of SMN itself have contributed greatly to our understanding of the cellular UsnRNP assembly machinery. Yet full-length SMN has so far evaded structural elucidation. The Seng et al. study claimed to have closed this gap, but based on the results presented here, the only conclusion that can be drawn is that the Seng et al. study is largely invalid and should be retracted from the literature., (© The Author 2016. Published by Oxford University Press.)

Phenol and its derivatives are common pollutants that are present in industrial discharge and are major xenobiotics that lead to water pollution. To monitor as well as improve water quality, attempts have been made in the past to engineer bacterial in vivo biosensors. However, due to the paucity of structural information, there is insufficiency in gauging the factors that lead to high sensitivity and selectivity, thereby impeding development. Here, we present the crystal structure of the sensor domain of MopR (MopR(AB)) from Acinetobacter calcoaceticus in complex with phenol and its derivatives to a maximum resolution of 2.5 Å. The structure reveals that the N-terminal residues 21-47 possess a unique fold, which are involved in stabilization of the biological dimer, and the central ligand binding domain belongs to the "nitric oxide signaling and golgi transport" fold, commonly present in eukaryotic proteins that bind long-chain fatty acids. In addition, MopR(AB) nests a zinc atom within a novel zinc binding motif, crucial for maintaining structural integrity. We propose that this motif is crucial for orchestrated motions associated with the formation of the effector binding pocket. Our studies reveal that residues W134 and H106 play an important role in ligand binding and are the key selectivity determinants. Furthermore, comparative analysis of MopR with XylR and DmpR sensor domains enabled the design of a MopR binding pocket that is competent in binding DmpR-specific ligands. Collectively, these findings pave way towards development of specific/broad based biosensors, which can act as useful tools for detection of this class of pollutants.

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme., (© 2016 The Protein Society.)

Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54(nrb)) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.

The Grb7 adaptor protein is a therapeutic target for both TNBC and HER2+ breast cancer. A nonphosphorylated cyclic peptide 1 (known as G7-18NATE) inhibits Grb7 via targeting the Grb7-SH2 domain, but requires the presence of phosphate ions for both affinity and specificity. Here we report the discovery of malonate bound in the phosphotyrosine binding pocket of the apo-Grb7-SH2 structure. Based on this, we carried out the rational design and synthesis of two analogues of peptide 1 that incorporate carboxymethylphenylalanine (cmF) and carboxyphenylalanine (cF) as mimics of phosphotyrosine (pY). Binding studies using SPR confirmed that affinity for Grb7-SH2 domain is improved up to 9-fold over peptide 1 under physiological phosphate conditions (KD = 2.1-5.7 μM) and that binding is specific for Grb7-SH2 over closely related domains (low or no detectable binding to Grb2-SH2 and Grb10-SH2). X-ray crystallographic structural analysis of the analogue bearing a cmF moiety in complex with Grb7-SH2 has identified the precise contacts conferred by the pY mimic that underpin this improved molecular interaction. Together this study identifies and characterizes the tightest specific inhibitor of Grb7 to date, representing a significant development toward a new Grb7-targeted therapeutic.

Using the UHV experimental endstation on the soft X-ray beamline at the Australian Synchrotron, lysozyme and proteinase K crystals have been exposed to a vacuum of 10 -5  mbar, prior to flash-cooling in a bath of liquid nitrogen. Subsequent data collection on the MX2 beamline at the Australian Synchrotron demonstrated that, for lysozyme and proteinase K, it is possible to subject these mounted crystals to a vacuum pressure of 10 -5  mbar without destroying the crystal lattice. Despite the lower data quality of the vacuum-pumped crystals compared with control crystals, it is demonstrated that the protein crystals can survive in a vacuum under suitable conditions.

Here, we review recent studies aimed at defining the importance of quaternary structure to a model oligomeric enzyme, dihydrodipicolinate synthase. This will illustrate the complementary and synergistic outcomes of coupling the techniques of analytical ultracentrifugation with enzyme kinetics, in vitro mutagenesis, macromolecular crystallography, small angle X-ray scattering, and molecular dynamics simulations, to demonstrate the role of subunit self-association in facilitating protein dynamics and enzyme function. This multitechnique approach has yielded new insights into the molecular evolution of protein quaternary structure., (© 2015 Elsevier Inc. All rights reserved.)

Aspartate kinase (AK) is an enzyme which is tightly regulated through feedback control and responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate. This intermediate step is at an important branch point where one path leads to the synthesis of lysine and the other to threonine, methionine and isoleucine. Concerted feedback inhibition of AK is mediated by threonine and lysine and varies between the species. The crystal structure of biotechnologically important Clostridium acetobutylicum aspartate kinase ( Ca AK; E.C. 2.7.2.4; Mw =48,030Da; 437aa; SwissProt: Q97MC0) has been determined to 3Å resolution. Ca AK acquires a protein fold similar to the other known structures of AKs despite the low sequence identity (<30%). It is composed of two domains: an N-terminal catalytic domain (kinase) domain and a C-terminal regulatory domain further comprised of two small domains belonging to the ACT domain family. Pairwise comparison of 12 molecules in the asymmetric unit helped to identify the bending regions which are in the vicinity of ATP binding site involved in domain movements between the catalytic and regulatory domains. All 12 Ca AK molecules adopt fully open T-state conformation leading to the formation of three tetramers unique among other similar AK structures. On the basis of comparative structural analysis, we discuss tetramer formation based on the large conformational changes in the catalytic domain associated with the lysine binding at the regulatory domains. The structure described herein is homologous to a target in wide-spread pathogenic (toxin producing) bacteria such as Clostridium tetani (64% sequence identity) suggesting the potential of the structure solved here to be applied for modeling drug interactions. Ca AK structure may serve as a guide to better understand and engineer lysine biosynthesis for the biotechnology industry.

The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties Lewis(B) and sialyl-Lewis(X), respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like β-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X) but not to Lewis(A), Lewis(B), or Lewis(Y). Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X). Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding.

Members of the WXG100 protein superfamily form homo- or heterodimeric complexes. The most studied proteins among them are the secreted T-cell antigens CFP-10 (10 kDa culture filtrate protein, EsxB) and ESAT-6 (6 kDa early secreted antigen target, EsxA) from Mycobacterium tuberculosis. They are encoded on an operon within a gene cluster, named as ESX-1, that encodes for the Type VII secretion system (T7SS). WXG100 proteins are secreted in a full-length form and it is known that they adopt a four-helix bundle structure. In the current work we discuss the evolutionary relationship between the homo- and heterodimeric WXG100 proteins, the basis of the oligomeric state and the key structural features of the conserved sequence pattern of WXG100 proteins. We performed an iterative bioinformatics analysis of the WXG100 protein superfamily and correlated this with the atomic structures of the representative WXG100 proteins. We find, firstly, that the WXG100 protein superfamily consists of three subfamilies: CFP-10-, ESAT-6- and sagEsxA-like proteins (EsxA proteins similar to that of Streptococcus agalactiae). Secondly, that the heterodimeric complexes probably evolved from a homodimeric precursor. Thirdly, that the genes of hetero-dimeric WXG100 proteins are always encoded in bi-cistronic operons and finally, by combining the sequence alignments with the X-ray data we identify a conserved C-terminal sequence pattern. The side chains of these conserved residues decorate the same side of the C-terminal α-helix and therefore form a distinct surface. Our results lead to a putatively extended T7SS secretion signal which combines two reported T7SS recognition characteristics: Firstly that the T7SS secretion signal is localized at the C-terminus of T7SS substrates and secondly that the conserved residues YxxxD/E are essential for T7SS activity. Furthermore, we propose that the specific α-helical surface formed by the conserved sequence pattern including YxxxD/E motif is a key component of T7SS-substrate recognition.

Dihydrodipicolinate synthase (DHDPS) catalyses the rate-limiting step in the biosynthesis of meso-diaminopimelate and lysine. Here, the cloning, expression, purification and crystallization of DHDPS from the intracellular pathogen Legionella pneumophila are described. Crystals grown in the presence of high-molecular-weight PEG precipitant and magnesium chloride were found to diffract beyond 1.65 Å resolution. The crystal lattice belonged to the hexagonal space group P6₁22, with unit-cell parameters a=b=89.31, c=290.18 Å, and contained two molecules in the asymmetric unit. The crystal structure was determined by molecular replacement using a single chain of Pseudomonas aeruginosa DHDPS as the search model.

The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity and potential role in human disease. These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic. The carboxy-terminal region of the C protein is the main cytotoxic component, and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells. Toxin complexes have been visualized using single-particle electron microscopy, but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a β-propeller domain, which mediates attachment of the B-C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example, to our knowledge, of a structure that contains rearrangement hotspot (RHS) repeats, and illustrates a marked structural architecture that is probably conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins. The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein encapsulation and delivery.

Plant HXXXD acyltransferase-catalyzed malonylation is an important modification reaction in elaborating the structural diversity of flavonoids and anthocyanins, and a universal adaptive mechanism to detoxify xenobiotics. Nicotiana tabacum malonyltransferase 1 (NtMaT1) is a member of anthocyanin acyltransferase subfamily that uses malonyl-CoA (MLC) as donor catalyzing transacylation in a range of flavonoid and naphthol glucosides. To gain insights into the molecular basis underlying its catalytic mechanism and versatile substrate specificity, we resolved the X-ray crystal structure of NtMaT1 to 3.1 Å resolution. The structure comprises two α/β mixed subdomains, as typically found in the HXXXD acyltransferases. The partial electron density map of malonyl-CoA allowed us to reliably dock the entire molecule into the solvent channel and subsequently define the binding sites for both donor and acceptor substrates. MLC bound to the NtMaT1 occupies one end of the long solvent channel between two subdomains. On superimposing and comparing the structure of NtMaT1 with that of an enzyme from anthocyanin acyltransferase subfamily from red chrysanthemum (Dm3Mat3) revealed large architectural variation in the binding sites, both for the acyl donor and for the acceptor, although their overall protein folds are structurally conserved. Consequently, the shape and the interactions of malonyl-CoA with the binding sites' amino acid residues differ substantially. These major local architectural disparities point to the independent, divergent evolution of plant HXXXD acyltransferases in different species. The structural flexibility of the enzyme and the amendable binding pattern of the substrates provide a basis for the evolution of the distinct, versatile substrate specificity of plant HXXXD acyltransferases.

In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e(-) reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.

Structural proteomics (SP) projects are capable of producing thousands of protein structures per year by employing semi-automated technologies. It is too early to assess and evaluate the scientific impact of these protein structures, although SP initiatives have substantially changed the traditional way of protein characterization. Many of the methodologies and technologies developed by SP have been adapted by structural biology laboratories and pharmaceutical companies to lower the costs, increase the speed and productivity of structure determination pipelines and to enhance drug discovery programs. The advent of genomic and proteomic technologies have facilitated rapid advances in our understanding of the molecular details of cellular function. The purpose of this review is to consider the impact of these technologies on protein structure analysis and to illustrate how it's directing the focus of research relevant to biotechnology.

Tuberculosis (TB) is a major global health threat caused by Mycobacterium tuberculosis (Mtb). It is further fueled by the HIV pandemic and by increasing incidences of multidrug resistant Mtb-strains. Rv2827c, a hypothetical protein from Mtb, has been implicated in the survival of Mtb in the macrophages of the host. The three-dimensional structure of Rv2827c has been determined by the three-wavelength anomalous diffraction technique using bromide-derivatized crystals and refined to a resolution of 1.93 A. The asymmetric unit of the orthorhombic crystals contains two independent protein molecules related by a non-crystallographic translation. The tertiary structure of Rv2827c comprises two domains: an N-terminal domain displaying a winged helix topology and a C-terminal domain, which appears to constitute a new and unique fold. Based on structural homology considerations and additional biochemical evidence, it could be established that Rv2827c is a DNA-binding protein. Once the understanding of the structure-function relationship of Rv2827c extends to the function of Rv2827c in vivo, new clues for the rational design of novel intervention strategies may be obtained.

Cutting carbons: The three-dimensional structure of polyneuridine aldehyde esterase (PNAE) gives insight into the enzymatic mechanism of the biosynthesis of C(9)- from C(10)-monoterpenoid indole alkaloids (see scheme). PNAE is a very substrate-specific serine esterase. It harbors the catalytic triad S87-D216-H244, and is a new member of the alpha/beta-fold hydrolase superfamily. Its novel function leads to the diversification of alkaloid structures.

The full-length LysR-type transcriptional regulator TsaR from Comamonas testosteroni T-2 was heterologously overexpressed in Escherichia coli, purified and stabilized under conditions that favoured its rapid crystallization using the microbatch-under-oil technique. The purified protein was highly crystallizable and two different crystal forms were readily obtained. However, only monoclinic crystals gave diffraction beyond 2 A and there was a slight variation in unit-cell parameters between crystals. The only other LysR-type regulator for which a full-length crystal form is available is CbnR, but no solution could be obtained when this was used as a model in molecular replacement. Mercury and xenon derivatives were therefore produced in order to phase the structure using a MIRAS approach.

The crystal structure of Escherichia coli ribonuclease I (EcRNase I) reveals an RNase T2-type fold consisting of a conserved core of six beta-strands and three alpha-helices. The overall architecture of the catalytic residues is very similar to the plant and fungal RNase T2 family members, but the perimeter surrounding the active site is characterized by structural elements specific for E. coli. In the structure of EcRNase I in complex with a substrate-mimicking decadeoxynucleotide d(CGCGATCGCG), we observe a cytosine bound in the B2 base binding site and mixed binding of thymine and guanine in the B1 base binding site. The active site residues His55, His133, and Glu129 interact with the phosphodiester linkage only through a set of water molecules. Residues forming the B2 base recognition site are well conserved among bacterial homologs and may generate limited base specificity. On the other hand, the B1 binding cleft acquires true base aspecificity by combining hydrophobic van der Waals contacts at its sides with a water-mediated hydrogen-bonding network at the bottom. This B1 base recognition site is highly variable among bacterial sequences and the observed interactions are unique to EcRNaseI and a few close relatives.

Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B.

Strictosidine beta-D-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of approximately 2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-207, Glu-416, His-161, and Trp-388 in catalysis. Comparison of the catalytic pocket of SG with that of other plant glucosidases demonstrates the structural importance of Trp-388. Compared with all other glucosidases of plant, bacterial, and archaeal origin, SG's residue Trp-388 is present in a unique structural conformation that is specific to the SG enzyme. In addition to STR1 and vinorine synthase, SG represents the third structural example of enzymes participating in the biosynthetic pathway of the Rauvolfia alkaloid ajmaline. The data presented here will contribute to deciphering the structure and reaction mechanism of other higher plant glucosidases.

The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of approximately 2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed beta-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed beta-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.

The temperate coliphage lambda, after infecting its host bacterium Escherichia coli, can develop either along the lytic or the lysogenic pathway. Crucial to the lysis/lysogeny decision is the homotetrameric transcription-activator protein CII (4 x 11 kDa) of the phage that binds to a unique direct-repeat sequence T-T-G-C-N6-T-T-G-C at each of the three phage promoters it activates: p(E), p(I), and p(aQ). Several regions of CII have been identified for its various functions (DNA binding, oligomerization, and susceptibility to host protease), but the crystal structure of the protein long remained elusive. Here, we present the three-dimensional structure of CII at 2.6-angstroms resolution. The CII monomer is comprised of four alpha helices and a disordered C terminus. The first three helices (alpha1-alpha3) form a compact domain, whereas the fourth helix (alpha4) protrudes in different orientations in each subunit. A four-helix bundle, formed by alpha4 from each subunit, holds the tetramer. The quaternary structure can be described as a dimer of dimers, but the tetramer does not exhibit a closed symmetry. This unusual quaternary arrangement allows the placement of the helix-turn-helix motifs of two of the four CII subunits for interaction with successive major grooves of B-DNA, from one face of DNA. This structure provides a simple explanation for how a homotetrameric protein may recognize a direct-repeat DNA sequence rather than the inverted-repeat sequences of most prokaryotic activators.