Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery.

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric linker." While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus.

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

Lanthanoid Heteroleptic Complexes with Cucurbit[5]uril and Dicarboxylate Ligands: From Discrete Structures to One-Dimensional and Two-Dimensional Polymers.

Lanthanoid heteroleptic complexes with cucurbit[5]uril {Q[5]} and two dicarboxylate ligands, e.g., diglycolic acid (H2DGC) and glutaric acid (H2GT), 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. [La4(Q[5])3(DGC)2(NO3)2(H2O)12][La(DGC)(H2O)6]·7NO3· nH2O (1) has a tetrametallic structure constructed with three bis-bidentate Q[5] ligands linking two [La(DGC)(H2O)2]+ species in the middle and two [La(H2O)4(NO3)]2+ species at both ends. [Ce2(Q[5])(DGC)(NO3)(H2O)10]·3NO3·4H2O (2) has a dimetallic structure built up with a bis-bidentate Q[5] ligand linking [Ce(DGC)(H2O)3(NO3)] and [Ce(H2O)7]3+ on each side of the Q[5] portals. [Ce3(Q[5])3(DGC)2(H2O)9][Ce(DGC)(H2O)6]2·7NO3· nH2O (3) has a 1D polymeric structure built up with bis-bidentate Q[5] ligands in-turn linking one [Ce(H2O)6]3+ and two [Ce(DGC)(H2O)6]1+ cationic species. [Ln2(Q[5])2(GT)(H2O)6]·4NO3· nH2O [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 GT2- ligands to form 2D polymers with a unique honeycomb-type topology. Their vibrational modes and electronic structures have also been investigated.

The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device.

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 ß-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.

Functional insights into the mode of DNA and ligand binding of the TetR family regulator TylP from Streptomyces fradiae.

Tetracycline repressors (TetRs) modulate multidrug efflux pathways in several pathogenic bacteria. In Streptomyces, they additionally regulate secondary metabolic pathways like antibiotic production. For instance, in the antibiotic producer Streptomyces fradiae, a layered network of TetRs regulates the levels of the commercially important antibiotic tylosin, with TylP occupying the top of this cascading network. TetRs exist in two functional states, the DNA-bound and the ligand-bound form, which are allosterically regulated. Here, to develop deeper insights into the factors that govern allostery, the crystal structure of TylP was solved to a resolution of 2.3 Å. The structure revealed that TylP possesses several unique features; notably, it harbors a unique C-terminal helix-loop extension that spans the entire length of the structure. This anchor connects the DNA-binding domain (DBD) with the ligand-binding domain (LBD) via a mix of positively charged and hydrogen-bonding interactions. Supporting EMSA studies with a series of ΔC truncated versions show that a systematic deletion of this region results in complete loss of DNA binding. The structure additionally revealed that TylP is markedly different in the orientation of its DBD and LBD architecture and the dimeric geometry from its hypothesized Streptomyces homologue CprB, which is a γ-butyrolactone regulator. Rather, TylP is closer in structural design to macrolide-binding TetRs found in pathogens. Supporting molecular dynamic studies suggested that TylP binds a macrolide intermediate in the tylosin pathway. Collectively, the structure along with corroborating biochemical studies provided insights into the novel mode of regulation of TetRs in antibiotic-producing organisms.

A critical examination of the recently reported crystal structures of the human SMN protein.

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.

MX2: a high-flux undulator microfocus beamline serving both the chemical and macromolecular crystallography communities at the Australian Synchrotron.

MX2 is an in-vacuum undulator-based crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range 4.8-21 keV to a focal spot of 22 × 12 µm FWHM (H × V). At 13 keV the flux at the sample is 3.4 × 1012 photons s-1. The beamline endstation allows robotic handling of cryogenic samples via an updated SSRL SAM robot. This beamline is ideal for weakly diffracting hard-to-crystallize proteins, virus particles, protein assemblies and nucleic acids as well as smaller molecules such as inorganic catalysts and organic drug molecules. The beamline is now mature and has enjoyed a full user program for the last nine years. This paper describes the beamline status, plans for its future and some recent scientific highlights.

Stonefish toxin defines an ancient branch of the perforin-like superfamily.

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and ß), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.

Interdomain Conformational Changes Provide Allosteric Regulation en Route to Chorismate.

Multifunctional proteins play a variety of roles in metabolism. Here, we examine the catalytic function of the combined 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated branch of the pathway for the biosynthesis of amino acids tyrosine and phenylalanine. In line with sequence predictions, the two catalytic functions are located in distinct domains, and these two activities can be separated and retain functionality. For the full-length protein, prephenate, the product of the CM reaction, acts as an allosteric inhibitor for the DAH7PS. The crystal structure of the full-length protein with prephenate bound and the accompanying small angle x-ray scattering data reveal the molecular mechanism of the allostery. Prephenate binding results in the tighter association between the dimeric CM domains and the tetrameric DAH7PS, occluding the active site and therefore disrupting DAH7PS function. Acquisition of a physical gating mechanism to control catalytic function through gene fusion appears to be a general mechanism for providing allostery for this enzyme.

Insights into ubiquitin-conjugating enzyme/ co-activator interactions from the structure of the Pex4p:Pex22p complex.

Ubiquitin-conjugating enzymes (E2s) coordinate distinct types of ubiquitination via specific E3 ligases, to a large number of protein substrates. While many E2 enzymes need only the presence of an E3 ligase for substrate ubiquitination, a number of E2s require additional, non-canonical binding partners to specify their function. Here, we have determined the crystal structure and function of an E2/co-activator assembly, the Pex4p:Pex22p complex. The peroxisome-associated E2 enzyme Pex4p binds the peroxisomal membrane protein Pex22p through a binding site that does not overlap with any other known interaction interface in E2 enzymes. Pex22p association enhances Pex4p's ability to transfer ubiquitin to a substrate in vitro, and Pex22p binding-deficient forms of Pex4p are unable to ubiquitinate the peroxisomal import receptor Pex5p in vivo. Our data demonstrate that the Pex4p:Pex22p assembly, and not Pex4p alone, functions as the E2 enzyme required for Pex5p ubiquitination, establishing a novel mechanism of E2 enzyme regulation.

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily.

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Šstructure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasi-racemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized d- and l-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.

The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding adhesin SabA from Helicobacter pylori.

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 ß-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.

MX1: a bending-magnet crystallography beamline serving both chemical and macromolecular crystallography communities at the Australian Synchrotron.

MX1 is a bending-magnet crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range from 8 to 18 keV to a focal spot at the sample position of 120 µm FWHM. The beamline endstation and ancillary equipment facilitate local and remote access for both chemical and biological macromolecular crystallography. Here, the design of the beamline and endstation are discussed. The beamline has enjoyed a full user program for the last seven years and scientific highlights from the user program are also presented.

The crystal structure of a homodimeric Pseudomonas glyoxalase†I enzyme reveals asymmetric metallation commensurate with half-of-sites activity.

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni(2+) ion, whereas the other contains two inactivating Zn(2+) ions. Enzymological experiments prompted by the binuclear Zn(2+) site identified a novel catalytic property of GloA2. The enzyme can function as a Zn(2+) /Co(2+) -dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction.

Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours.

Insight into the structure and inhibition mechanism of O-ß-d-glucosidases by deoxa-pyranosylamine type inhibitors is provided by X-ray analysis of complexes between raucaffricine and strictosidine glucosidases and N-(cyclohexylmethyl)-, N-(cyclohexyl)- and N-(bromobenzyl)-ß-d-gluco-1,5-deoxa-pyranosylamine. All inhibitors anchored exclusively in the catalytic active site by competition with appropriate enzyme substrates. Thus facilitated prospective elucidation of the binding networks with residues located at <3.9 Å distance will enable the development of potent inhibitors suitable for the production of valuable alkaloid glucosides, raucaffricine and strictosidine, by means of synthesis in Rauvolfia serpentina cell suspension cultures.

Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases.

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.

Cyanuric acid hydrolase: evolutionary innovation by structural concatenation.

The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the 'Toblerone' fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser-Lys catalytic dyads. A single catalytic dyad (Ser85-Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure-function relationship.

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins.

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

In-house UV radiation-damage-induced phasing of selenomethionine-labeled protein structures.

Selenomethionine labeling is the most common technique used in protein crystallography to derivatize recombinant proteins for experimental phasing using anomalous scattering at tunable synchrotron beamlines. Recently, it has been shown that UV radiation depletes electron density of selenium atoms of selenomethionine residues and that UV radiation-damage-induced phasing (equivalent to single isomorphous replacement) protocol can be applied to calculate experimental phases. Here we present the straightforward integration of a UV source with an in-house diffractometer. We show how this setup can extend the capabilities of a sealed tube X-ray generator and be used for experimental phasing of selenium-labeled proteins.

Crystal structure of perakine reductase, founding member of a novel aldo-keto reductase (AKR) subfamily that undergoes unique conformational changes during NADPH binding.

Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His(6)-PR was solved to 2.31 Å. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His(6)-PR-A213W complex with NADPH was determined at 1.77 Å. Overall, PR folds in an unusual α(8)/ß(6) barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional ß-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 Å occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family.