Structural characterization of a short-chain dehydrogenase/reductase from multi-drug resistant Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) is a clinically relevant, highly drug-resistant pathogen of global concern. An attractive approach to drug design is to specifically target the type II fatty acid synthesis (FASII) pathway which is critical in Gram negative bacteria and is significantly different to the type I fatty acid synthesis (FASI) pathway found in mammals. Enzymes involved in FASII include members of the short-chain dehydrogenase/reductase (SDR) superfamily. SDRs are capable of performing a diverse range of biochemical reactions against a broad spectrum of substrates whilst maintaining conserved structural features and sequence motifs. Here, we use X-ray crystallography to describe the structure of an SDR from the multi-drug resistant bacteria A. baumannii, previously annotated as a putative FASII FabG enzyme. The protein was recombinantly expressed, purified, and crystallized. The protein crystals diffracted to 2.0 Šand the structure revealed a FabG-like fold. Functional assays revealed, however, that the protein was not active against the FabG substrate, acetoacetyl-CoA. This study highlights that database annotations may show the necessary structural hallmarks of such proteins, however, they may not be able to cleave substrates that are typical of FabG enzymes. These results are important for the selection of target enzymes in future drug development.

Crystal structure of the integral membrane diacylglycerol kinase.

Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology, folding, assembly and stability. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

Structure of the NDH-2 - HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors.

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ1) interactions in the yeast Ndi1-UQ1 complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn24 and Asp39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase.

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.

Structural insights into electron transfer in caa3-type cytochrome oxidase.

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.

Mycobacteria Encode Active and Inactive Classes of TesB Fatty-Acyl CoA Thioesterases Revealed through Structural and Functional Analysis.

Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.

Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism.

PaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn(37), Asp(52), and Thr(68) are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N terminus, and a loop region involving Tyr(38)-Tyr(39). This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.

Structure and function of an irreversible agonist-ß(2) adrenoceptor complex.

G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human ß(2) adrenergic receptor (ß(2)AR) as a guide, we designed a ß(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent ß(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound ß(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 µs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

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.

Operation of the Australian Store.Synchrotron for macromolecular crystallography.

The Store.Synchrotron service, a fully functional, cloud computing-based solution to raw X-ray data archiving and dissemination at the Australian Synchrotron, is described. The service automatically receives and archives raw diffraction data, related metadata and preliminary results of automated data-processing workflows. Data are able to be shared with collaborators and opened to the public. In the nine months since its deployment in August 2013, the service has handled over 22.4 TB of raw data (∼1.7 million diffraction images). Several real examples from the Australian crystallographic community are described that illustrate the advantages of the approach, which include real-time online data access and fully redundant, secure storage. Discoveries in biological sciences increasingly require multidisciplinary approaches. With this in mind, Store.Synchrotron has been developed as a component within a greater service that can combine data from other instruments at the Australian Synchrotron, as well as instruments at the Australian neutron source ANSTO. It is therefore envisaged that this will serve as a model implementation of raw data archiving and dissemination within the structural biology research community.

Structural determinants of phosphorylation-dependent nuclear transport of HCMV DNA polymerase processivity factor UL44.

Human cytomegalovirus DNA polymerase processivity factor UL44 is transported into the nucleus by importin (IMP) α/ß through a classical nuclear localization signal (NLS), and this region is susceptible to cdc2-mediated phosphorylation at position T427. Whilst phosphorylation within and close to the UL44 NLS regulates nuclear transport, the details remain elusive, due to the paucity of structural information regarding the role of negatively charged cargo phosphate groups. We addressed this issue by studying the effect of UL44 T427 phosphorylation on interaction with several IMPα isoforms by biochemical and structural approaches. Phosphorylation decreased UL44/IMPα affinity 10-fold, and a comparative structural analysis of UL44 NLS phosphorylated and non-phosphorylated peptides complexed with mouse IMPα2 revealed the structural rearrangements responsible for phosphorylation-dependent inhibition of UL44 nuclear import.

Structural characterisation of a MAPR-related archaeal cytochrome b5M protein.

We recently reported that the membrane-associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b5 (cytb5 ) domain proteins, called cytb5M (MAPR-like). Relative to classical cytb5 proteins, MAPR and ctyb5M proteins shared unique sequence elements and a distinct heme-binding orientation at an approximately 90° rotation relative to classical cytb5 , as demonstrated in the archetypal crystal structure of a cytb5M protein (PDB accession number 6NZX). Here, we present the crystal structure of an archaeal cytb5M domain (Methanococcoides burtonii WP_011499504.1, PDB:6VZ6). It exhibits similar heme binding to the 6NZX cytb5M , supporting the deduction that MAPR-like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene.

MERS-CoV ORF4b employs an unusual binding mechanism to target IMPα and block innate immunity.

The MERS coronavirus (MERS-CoV) is a highly pathogenic, emerging virus that produces accessory proteins to antagonize the host innate immune response. The MERS-CoV ORF4b protein has been shown to bind preferentially to the nuclear import adapter IMPα3 in infected cells, thereby inhibiting NF-κB-dependent innate immune responses. Here, we report high-resolution structures of ORF4b bound to two distinct IMPα family members. Each exhibit highly similar binding mechanisms that, in both cases, lack a prototypical Lys bound at their P2 site. Mutations within the NLS region dramatically alter the mechanism of binding, which reverts to the canonical P2 Lys binding mechanism. Mutational studies confirm that the novel binding mechanism is important for its nuclear import, IMPα interaction, and inhibition of innate immune signaling pathways. In parallel, we determined structures of the nuclear binding domain of NF-κB component p50 bound to both IMPα2 and α3, demonstrating that p50 overlaps with the ORF4b binding sites, suggesting a basis for inhibition. Our results provide a detailed structural basis that explains how a virus can target the IMPα nuclear import adapter to impair immunity, and illustrate how small mutations in ORF4b, like those found in closely related coronaviruses such as HKU5, change the IMPα binding mechanism.

Insights into Acinetobacter baumannii fatty acid synthesis 3-oxoacyl-ACP reductases.

Treatments for 'superbug' infections are the focus for innovative research, as drug resistance threatens human health and medical practices globally. In particular, Acinetobacter baumannii (Ab) infections are repeatedly reported as difficult to treat due to increasing antibiotic resistance. Therefore, there is increasing need to identify novel targets in the development of different antimicrobials. Of particular interest is fatty acid synthesis, vital for the formation of phospholipids, lipopolysaccharides/lipooligosaccharides, and lipoproteins of Gram-negative envelopes. The bacterial type II fatty acid synthesis (FASII) pathway is an attractive target for the development of inhibitors and is particularly favourable due to the differences from mammalian type I fatty acid synthesis. Discrete enzymes in this pathway include two reductase enzymes: 3-oxoacyl-acyl carrier protein (ACP) reductase (FabG) and enoyl-ACP reductase (FabI). Here, we investigate annotated FabG homologs, finding a low-molecular weight 3-oxoacyl-ACP reductase, as the most likely FASII FabG candidate, and high-molecular weight 3-oxoacyl-ACP reductase (HMwFabG), showing differences in structure and coenzyme preference. To date, this is the second bacterial high-molecular weight FabG structurally characterized, following FabG4 from Mycobacterium. We show that ΔAbHMwfabG is impaired for growth in nutrient rich media and pellicle formation. We also modelled a third 3-oxoacyl-ACP reductase, which we annotated as AbSDR. Despite containing residues for catalysis and the ACP coordinating motif, biochemical analyses showed limited activity against an acetoacetyl-CoA substrate in vitro. Inhibitors designed to target FabG proteins and thus prevent fatty acid synthesis may provide a platform for use against multidrug-resistant pathogens including A. baumannii.

Structural basis for nuclear import selectivity of pioneer transcription factor SOX2.

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.

Crystallizing transmembrane peptides in lipidic mesophases.

Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not apply to small protein targets having

Crystal structure of posnjakite formed in the first crystal water-cooling line of the ANSTO Melbourne Australian Synchrotron MX1 Double Crystal Monochromator.

Exceptionally large crystals of posnjakite, Cu4SO4(OH)6(H2O), formed during corrosion of a Swagelock(tm) Snubber copper gasket within the MX1 beamline at the ANSTO-Melbourne, Australian Synchrotron. The crystal structure was solved using synchrotron radiation to R 1 = 0.029 and revealed a structure based upon [Cu4(OH)6(H2O)O] sheets, which contain Jahn-Teller-distorted Cu octa-hedra. The sulfate tetra-hedra are bonded to one side of the sheet via corner sharing and linked to successive sheets via extensive hydrogen bonds. The sulfate tetra-hedra are split and rotated, which enables additional hydrogen bonds.

The structure and activity of the glutathione reductase from Streptococcus pneumoniae.

The glutathione reductase (GR) from Streptococcus pneumoniae is a flavoenzyme that catalyzes the reduction of oxidized glutathione (GSSG) to its reduced form (GSH) in the cytoplasm of this bacterium. The maintenance of an intracellular pool of GSH is critical for the detoxification of reactive oxygen and nitrogen species and for intracellular metal tolerance to ions such as zinc. Here, S. pneumoniae GR (SpGR) was overexpressed and purified and its crystal structure determined at 2.56 Šresolution. SpGR shows overall structural similarity to other characterized GRs, with a dimeric structure that includes an antiparallel ß-sheet at the dimer interface. This observation, in conjunction with comparisons with the interface structures of other GR enzymes, allows the classification of these enzymes into three classes. Analyses of the kinetic properties of SpGR revealed a significantly higher value for Km(GSSG) (231.2 ± 24.7 µM) in comparison to other characterized GR enzymes.

Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6.

Assembly factors play key roles in the biogenesis of many multi-subunit protein complexes regulating their stability, activity, and the incorporation of essential cofactors. The human assembly factor Coa6 participates in the biogenesis of the CuA site in complex IV (cytochrome c oxidase, COX). Patients with mutations in Coa6 suffer from mitochondrial disease due to complex IV deficiency. Here, we present the crystal structures of human Coa6 and the pathogenic W59CCoa6-mutant protein. These structures show that Coa6 has a 3-helical bundle structure, with the first 2 helices tethered by disulfide bonds, one of which likely provides the copper-binding site. Disulfide-mediated oligomerization of the W59CCoa6 protein provides a structural explanation for the loss-of-function mutation.