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Automatic Processing of Rotation Diffraction Data from Crystals of Initially Unknown Symmetry and Cell Constants
Journal of Applied Crystallography
Abstract
An algorithm has been developed for the automatic interpretation of a given set of observed reciprocal-lattice points. It extracts a reduced cell and assigns indices to each reflection by a graph-theoretical implementation of the local indexing method. All possible symmetries of the observed lattice compatible with the metric of the reduced cell are recognized and reported, together with the unit-cell constants and the linear index transformation relating the conventional to the reduced cell. This algorithm has been incorporated into the program XDS [Kabsch (1988). J. Appl. Cryst.21, 916–924], which is now able to process single-crystal area-detector data without prior knowledge of the symmetry and the unit-cell constants.
... Diffraction data were collected at the X12 beamline of DESY (Hamburg, Germany) equipped with a MAR CCD 165 detector. The data were processed and merged with the XDS package [18] (version May 8, 2007). Analysis of the results showed that the P3 1 and P3 1 21 space groups were the most probable. ...
... Though the protein was cocrystallized with the 10-fold molar excess of the GTP analog GDPCP, as in all previously solved structures of nucleotide-bound SsoIF2γ [9,10], no nucleotide was found in the structure. The model demonstrates the apo form of the protein, and similar to other apo forms of SsoIF2γ the nucleotide-binding pocket is occupied by amino acid residues (18)(19)(20) of the P-loop (Fig. 1). ...
The crystal structure of the γ-subunit of translation initiation factor 2 from the archaeon Sulfolobus solfataricus
(SsoIF2γ) has been solved based on perfectly hemihedral twinned data. The protein was cocrystallized with the 10-fold
molar excess of GTP analog (GDPCP) over protein. However, no nucleotide was found in the structure, and the model
demonstrated the apo form of the protein. Two slightly different molecules in the asymmetric unit of the crystal are related
by the non-crystallographic 2-fold axis and form a tightly associated dimer. This dimer is stabilized by an intermolecular
hydrophobic core and hydrogen bonds. Lack of GDPCP in the nucleotide-binding pocket of the γ-subunit and significant
excess of dimers over monomers in the crystallization solution suggest that these dimers are the building blocks of the crystal. Contrary to SsoIF2γ monomers, these dimers are able to crystallize in two oppositely oriented slightly different crystal
domains, thus forming a twinned crystal. Comparison of crystallization conditions for the twinned and untwinned crystals
of apo SsoIF2γ showed that stabilization of the dimers in the solution may be caused by higher sodium salt concentration.
Since amino acid residues involved in intermolecular contacts in the dimer are responsible for binding of the γ- and α-subunits within SsoIF2, increase in sodium salt concentration may prevent functioning of SsoIF2 in the cell.
... Diffraction data were collected using the Advanced Photon Source (Argonne, Illinois, USA) with various beamlines (SI Appendix, Table S3). Data were processed using XDS (Build January 26, 2018) and molecular replacement; refinement was done using Molrep and Refmac within CCP4 (Version 7.0) and Coot (v0.8.9) (55)(56)(57)(58)(59). For the molecular replacement which was done similarly by a case study described by McCoy et al. (60), two separate models from the AlphaFold database were used (both 93% seq. ...
Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug’s direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition ( mfmA and mfmB ) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (k cat /K M ) of 9.6 × 10 ³ M ⁻¹ s ⁻¹ and K M for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.
... Data sets were collected at the Advanced Photon Source 24 ID-C synchrotron facility to a maximum resolution of 1.57 Å. Raw diffraction data were processed using XDS ( 29 ), Pointless ( 30 ), and Aimless ( 31 ). The structure was solved via molecular replacement (MR) using Phenix.Phaser (version 1.13_2998) ( 32 ). ...
Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5′-ATGGA ATGGA ATGGA ATGGA-3′ using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05–0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G–G intercalation sandwiched by sheared A–G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A–T/T–A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.
... X-ray diffraction data were collected on beamline ID30A-1 (Massif-1) at ESRF, Grenoble, France. The dataset was processed by the autoPROC pipeline for 1 (Vonrhein et al, 2011) and using XDS for 27 (Kabsch, 1993) (Table S2). ...
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC 50 of 14 µM. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC 50 of 2.1 µM and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of a mouse hepatitis virus, a prototype CoV. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.
... It is made processed using XDS. 23 The structures were determined by molecular replacement with PHASER using DosS (PDB ID: 3ZXO) as the search model. 16,[24][25][26][27] Iterative model building and refinement were conducted using COOT and PHENIX. ...
DosS is a heme-sensor histidine kinase that responds to redox-active stimuli in mycobacterial environments by triggering dormancy transformation. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases suggests that it possesses a rather short ATP-lid. This feature has been thought to inhibit DosS kinase activity by blocking ATP binding in the absence of interdomain interactions with the dimerization and histidine phospho-transfer (DHp) domain of full-length DosS. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS’s CA domain. We show that the closed lid conformation observed in protein crystal structures of DosS CA is caused by the presence of a zinc cation in the ATP binding pocket that coordinates with a glutamate residue on the ATP-lid. Furthermore, circular dichroism (CD) studies and comparisons of DosS CA crystal structure with its AlphaFold model and homologous DesK reveal that a key N-box alpha-helix turn of the ATP pocket manifests as a random coil in the zinc-coordinated protein crystal structure. We note that this closed lid conformation and the random-coil transformation of an N-box alpha-helix turn are artifacts arising from the millimolar zinc concentration used in DosS CA crystallization conditions. In contrast, in the absence of zinc, we find that the short ATP-lid of DosS CA has significant conformational flexibility and can bind ATP ( K d = 53 ± 13 μM). We conclude that DosS CA is almost always bound to ATP under physiological conditions (1-5 mM ATP, sub-nanomolar free zinc) in the bacterial environment. Our findings elucidate the conformational adaptability of the short ATP-lid, its relevance to ATP binding in DosS CA and provide insights that extends to 2988 homologous bacterial proteins containing such ATP-lids.
... X-ray data were collected at beamline BL14-1 at the BESSY synchrotron radiation facility (Berlin, Germany) [75]. Diffraction data were collected to a resolution of about 1.9 Å and processed using the XDS/XDSAPP program package [76,77] (electronic supplementary material, figure S1A and table S1). The molecular replacement software Phaser placed four NTF2 monomers into the asymmetric unit (asu) royalsocietypublishing.org/journal/rsob Open Biol. ...
G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.
... Crystals were flash frozen in reservoir solution supplemented with glycerol 15% (v/v). Data were collected at 100˚K at PROXIMA 1 beamline of the synchrotron SOLEIL and processed using XDS [67] and AIMLESS [68] from the CCP4 program suite [69,70]. Crystals of CagI:K5 and CagI:K2 diffracted to resolutions of 2.0 Å and 1.8 Å, respectively and belonged to the orthorhombic space group P2 1 2 1 2 1 with very similar cell dimensions ( Table 2). ...
The bacterial human pathogen Helicobacter pylori produces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. The cagT4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shape N-terminal domains (CagIN) prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagIC with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagIC were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagIC plays a key role in cagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cagT4SS, a crucial risk factor for gastric cancer development.
... Using the software package XDS [27], data reduction was performed for indexing, Lorentz-polarization correction, integration, scaling, and cell parameter refinements. The obtained hkl files were corrected for oblique phosphor incidence using the program PHOSCOR [28]. ...
This study explains the ionic conductivity in the mineral sugilite (idealized formula: Fe2Na2K[Li3Si12O30]) by resolving the dynamic disorder of both Li and Na cations using synchrotron X-ray single-crystal diffraction from 298 K to 1023 K. Non-zero anharmonic atomic displacement parameters at Na and Li sites at 1023 K adumbrated long-range charge transport routes for Li and Na cations commonly parallel to the (a–b) plane. Temperature-enhanced diffuse residuals in Fourier maps could unambiguously localize two interstitial sites suitable for Li, as well as three for Na. Each two-dimensional (2D) network of Li and Na interstitials was formed parallel to each other, providing Li and Na hopping pathways. The higher concentration of Na cations hopping in short distances of 2.0962(4)–2.3015(5) Å could be the main reason for the higher bulk conductivity values evaluated by impedance spectra of sugilite in comparison to those of its structural relatives with low Na contents, e.g., the mineral sogdianite ((Zr,Al,Fe)2Na0.36K[Li3Si12O30]). Bond valence sum landscape maps supported the critical role of dynamic disorder of Na+ over densely packed 2D interstitial networks for combined ionic conductivity along with mobile Li+ in sugilite-type compounds.
... A focused beam with a FWHM size of 7 × 3 µm, at an energy of 12.7 keV (0.9763 Å) at a flux of~4 × 10 11 ph/s and an exposure time of 7.5 ms per image was used during data-collection on an Eiger2 CdTe 16 M detector for P14 and the Eiger16M for P13 (Dectris, Switzerland). Diffraction data were processed using XDS [29][30][31] and AutoPROC using StarAniso 32,33 . For processing the TS datasets, the collected datasets were initially integrated using XDS and merged and scaled using the CCP4 suite program AIMLESS 34,35 . ...
We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.
... A complete MAD data set at four wavelengths was collected in order to solve the crystal structure of VirD. Data sets were indexed and integrated using XDS 51 and scaled by using pointless and aimless (CCP4 package). ...
During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, variations in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.
... The K161Q diffraction pattern was processed using XDS [47][48][49][50][51][52][53][54][55] and solved by molecular replacement 56 using the structure of wild-type Ack1 (Protein Data Bank, PDB: 4EWH) as a search model. The Ack1 K161Q model was built and refined using Coot 57 and PHENIX, 58 respectively. ...
Tyrosine kinases (TKs) play essential roles in signaling processes that regulate cell survival, migration, and proliferation. Dysregulation of tyrosine kinases underlies many disorders, including cancer, cardiovascular and developmental diseases, as well as pathologies of the immune system. Ack1 and Brk are nonreceptor tyrosine kinases (NRTKs) best known for their roles in cancer. Here, we have biochemically characterized novel Ack1 and Brk mutations identified in patients with systemic lupus erythematosus (SLE). These mutations are the first SLE-linked polymorphisms found among NRTKs. We show that two of the mutants are catalytically inactive, while the other three have reduced activity. To understand the structural changes associated with the loss-of-function phenotype, we solved the crystal structure of one of the Ack1 kinase mutants, K161Q. Furthermore, two of the mutated residues (Ack1 A156 and K161) critical for catalytic activity are highly conserved among other TKs, and their substitution in other members of the kinase family could have implications in cancer. In contrast to canonical gain-of-function mutations in TKs observed in many cancers, we report loss-of-function mutations in Ack1 and Brk, highlighting the complexity of TK involvement in human diseases.
... After generating a sufficient number of samples, we cluster them using the K-means [36] algorithm with 2N ref clusters, then pick the center of each cluster as outputs. In clustering, we first align and centralize those 3D coordinates by kabsch algorithm [37], then simply apply K-means on the flattened vector of aligned coordinates. In this way, we obtain twice as many conformations as N ref for metric calculations, following the previous setting [18,20]. ...
Molecular conformation generation (MCG) is a fundamental and important problem in drug discovery. Many traditional methods have been developed to solve the MCG problem, such as systematic searching, model-building, random searching, distance geometry, molecular dynamics, Monte Carlo methods, etc. However, they have some limitations depending on the molecular structures. Recently, there are plenty of deep learning based MCG methods, which claim they largely outperform the traditional methods. However, to our surprise, we design a simple and cheap algorithm (parameter-free) based on the traditional methods and find it is comparable to or even outperforms deep learning based MCG methods in the widely used GEOM-QM9 and GEOM-Drugs benchmarks. In particular, our design algorithm is simply the clustering of the RDKIT-generated conformations. We hope our findings can help the community to revise the deep learning methods for MCG. The code of the proposed algorithm could be found at https://gist.github.com/ZhouGengmo/5b565f51adafcd911c0bc115b2ef027c.
... Single Crystal X-ray Diffraction (SCXRD) data were collected for 7, 9 and 20 at the Australian Synchrotron MX2 beamline [1] at 100(2) K using a wavelength of λ = 0.7108 Å. Synchrotron data acquisition was carried out using AS QEGUI [2] and subsequent data indexation, reduction and integration was performed with XDS. [3] Data for 18 were collected using a RigakuOD XtaLAB Synergy at 150(2) K with micro-focused MoKα radiation (λ = 0.71073) Å). Acquisition, indexation, reduction and integration for 18 were performed with the CrysAlis software package [4] and a multi-scan absorption correction applied through the use of spherical harmonics, implemented in the SCALE 3 ABSPACK scaling algorithm. ...
Neurodegenerative diseases impose a considerable medical and public health burden on populations throughout the world. Oxidative stress, an imbalance in pro-oxidant/antioxidant homeostasis that leads to the generation of reactive oxygen species (ROS), has been implicated in the progression of a number of neurodegenerative diseases. The manipulation of ROS levels may represent a promising treatment option to slow down neurodegeneration, although adequate potency of treatments has not yet been achieved. Using a hybrid pharmacology approach, free radical nitroxide antioxidants were hybridised with a class of natural antioxidants, flavonoids, to form a potential multitargeted antioxidant. Modification of the Baker-Venkataraman reaction achieved the flavonoid-nitroxide hybrids (6-9) in modest yields. Antioxidant evaluation of the hybrids by cyclic voltammetry showed both redox functionalities were still active, with little influence on oxidation potential. Assessment of the peroxyl radical scavenging ability through an ORAC assay showed reduced antioxidant activity of the hybrids compared to their individual components. It was hypothesized that the presence of the phenol in the hybrids creates a more acidic medium which does not favour regeneration of the nitroxide from the corresponding oxammonium cation, disturbing the typical catalytic cycle of peroxyl radical scavenging by nitroxides. This work highlights the potential intricacies involved with drug hybridization as a strategy for new therapeutic development.
... X-ray crystallography: TMU-82 was measured at the Australian Synchrotron on the MX1 macromolecular beamline, data integration was completed using XDS [2] software programs. ...
Extensive research over the past few years has been focused on the synthesis of MOFs with high internal surface areas, however, introducing functional groups on their backbones at the same...
... Data were collected at SOLEIL on beamline Proxima-2 from a single crystal that diffracted up to 3.7 Å resolution and belonged to the space group P6 2 22, with 2 molecules per asymmetric unit. Diffraction data were processed using XDS 61 and with AIMLESS 62 from the CCP4 programme suite 63 . Data collection statistics are indicated in Supplementary Table 2. ...
The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen. The bacterium Brucella abortus is an intracellular pathogen that modulates autophagy in host cells. Here, the authors identify two B. abortus effectors that interact with host protease SENP3, thus promoting cytoplasmic accumulation of nucleolar proteins associated with ribosomal biogenesis and facilitating intracellular replication of the pathogen
... Single crystals of complexes [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-HBH) 2 ]Cl 2 ⋅2CH 3 CN, and [PdCl(DPPB-PTSC)] were dipped into a viscous hydrocarbon oil and mounted onto a loop to determine their structures using Australian Synchrotron over MX1 macromolecular beamlines at 100 K and λ = 0.71073 Å. All diffractions were indexed and integrated by the XDS software suite [30]. SHELXT was applied to solve data refining against F 2 via the full-matrix least-squares method with SHELXL-2018 in Olex2 [31,32] ...
Herein we studied the synthesis and the biological properties of palladium (II) complexes derived from iminophosphine ligands DPPB-3-hydroxybenzohydrazone (DPPB-HBH), DPPB-4-phenylthiosemicarbazone (DPPB-PTSC), and DPPB-thiosemicarbazone (DPPB-TSC)
(DPPB = 2-(diphenylphosphino)benzaldehyde) using theoretical and experimental methods. The molecular structures of [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-HBH)2]2Cl∙2CH3CN, [PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)] were characterized by various spectroscopic techniques and X-ray crystallographic for [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-HBH)2]2Cl∙2CH3CN, and [PdCl(DPPB-PTSC)], confirming the distorted square-planar geometry for the palladium atoms. Complexes with the general formula [PdCl(Ligand)x] (x = 1) were employed to evaluate biological activities such as cytotoxicity against the MDA-MB-231 breast cancer cells and DNA binding ability. The results revealed the high cytotoxicity of the palladium complexes compared to the reference drug cisplatin (IC50= 24.59 µg/mL), as well as their moderate binding ability to DNA through electrostatic interactions and groove binding. It was also found that [PdCl(DPPB-HBH)]Cl has the highest activity among others (IC50 = 9.3 µg/mL). Their interaction mode with DNA was investigated using molecular docking and NCI calculations, which showed complete agreement with the experimental results. Furthermore, quantum chemistry calculations were used to analyze the structure-activity relationship and introduce reactive sites, which determined the cause of the difference in the reactivity of the samples.
... Crystals were harvested, cryo-protected in a reservoir solution supplemented with 25% glycerol and flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K at the Beamline station 4.2.2 at the Advanced Light Source (Berkeley National Laboratory, CA) and were initially indexed, integrated, and scaled using XDS 82 . Molecular replacement was used to estimate phases using PHASER and PDB code 4Z7E 38 as search model. ...
L -Ergothioneine (ET), the 2-thioimidazole derivative of trimethylhistidine, is biosynthesized by select fungi and bacteria, notably Mycobacterium tuberculosis , and functions as a scavenger of reactive oxygen species. The extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd _1642-1643 in Streptococcus pneumoniae , a Gram-positive respiratory pathogen, encodes an ET uptake ATP-binding cassette (ABC) transporter, designated EgtU. The solute binding domain (SBD) of EgtU, EgtUC, binds ET with high affinity and exquisite specificity in a cleft between the two subdomains, with cation-π interactions engaging the betaine moiety and a network of water molecules that surround the thioimidazole ring. EgtU is highly conserved among known quaternary amine compound-specific transporters and widely distributed in Firmicutes, including the human pathogens Listeria monocytogenes , as BilEB, Enterococcus faecalis and Staphylococcus aureus . ET increases the chemical diversity of the low molecular weight thiol pool in Gram-positive human pathogens and may contribute to antioxidant defenses in the infected host.
... X-ray crystallography data collection, structure solution, and refinement. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF), France at beamlines ID23-1 and BM30A (currently called BM07) and processed with XDS (59). Structures were solved by molecular replacement using previously published RHDV GI.2 P domain (PDB ID: 4X1W) and Fab fragment structures (PDB ID: 3V7A) as search models in PHASER (60). ...
Isolated RHDV antibodies have been used for decades to distinguish between antigenic variants, monitor temporal capsid evolution, and examine neutralizing capacities. In this study, we provided the structural basis for an RHDV GI.2 specific diagnostic antibody (2D9) binding and reveal that a small number of amino acid substitutions at the binding site could differentiate between RHDV GI.2 and GI.1b.
... The crystals were then flash-frozen in a nitrogen gas stream at 100 K. X-ray diffraction images were collected at the beamline BL41XU of SPring-8, Japan. The dataset obtained was indexed, integrated, and scaled with XDS (59). The initial phase up to 4 Å resolution was obtained by molecular replacement with PaserMR in CCP4 program suite (60) using the 1.90 Å resolution structure of native PSII (PDB accession code: 3WU2) as the search model, and both structures were refined to 1.90 Å resolution with Refmac5 of CCP4 program suite (61) and Phenix refinement (62). ...
Three psbA genes (psbA1, psbA2, and psbA3) that encode for a key photosystem II (PSII) subunit are present in the thermophilic cyanobacterium Thermosynechococcus elongatus, and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen-bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to the change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen-bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, and we found the channel to be narrower, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen-bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, which may explain the oxygen evolution enhancement in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.
... The data were indexed using XDS and then integrated using Mosflm (Leslie, 1992). The data were finally scaled using XDS (Kabsch, 1993) (Table 2.3). ...
MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium. Initial investigation into this enzyme categorized it as a methyltransferase. Further investigation revealed that this was incorrect, and that MosA is actually a dihydrodipicolinate synthase, part of the N-acetylneuraminate lyase superfamily. One of the characteristics of enzyme superfamilies is their low sequence identity, but relatively high structural similarity. The structural investigation reported here confirms the high structural similarity between MosA and other superfamily members.
Investigation of MosA was carried out by means of x-ray crystallography. It was believed that detailed structural information may shed light into not only the enzymatic mechanism, but also the inhibition of MosA by lysine, the final product of the enzymatic pathway. Insight into enzyme mechanism and inhibition may ultimately prove useful in herbicide or insecticide development, as other dihydrodipicolinate synthases from harmful fungi, bacteria, or plants, make attractive targets for inhibition. Lysine is an essential amino acid for humans, meaning that there is no endogenous lysine production to block the use of these hypothetical inhibitors. Specific inhibitors based on crystal structures have proven to be effective in the past and hopefully, will continue to be useful in the future.
Here we report the structure of MosA, solved to 1.95 Å resolution with lysine 161 forming a Schiff-base adduct with pyruvate. This adduct is consistent with the currently accepted dihydrodipicolinate synthase enzyme mechanism.
... A complete MAD data set at four wavelengths was collected in order to solve the crystal structure of VirD. Data sets were indexed and integrated using XDS 44 and scaled by using pointless and aimless (CCP4 package). ...
During biosynthesis by multi-modular trans-AT polyketide synthases (PKSs), polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase (HMGS) cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT PKS. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation, identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, imperfections in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.
... Single crystal data was collected on the MX1 beamline at the Australian Synchrotron, using silicon double crystal monochromatic radiation (λ = 0.71073 Å) at 100 K [15]. The XDS software package [16] was used on site for data integration, processing and scaling. SADABS [17] was used to apply an empirical absorption correction. ...
Inspired by ethnopharmacological knowledge, we conducted a bioassay-guided fractionation of the leaves of Tristaniopsis laurina which led to the discovery of a new anti-inflammatory compound, tristaenone A (1). The structure was elucidated by detailed spectroscopic data analysis, and the absolute configuration was established using X-ray crystallography analysis. Tristaenone A (1) suppressed LPS and IFN-γ-induced NO, TNF-α and IL-6 production in RAW 264.7 cells with IC50 values of 37.58 ± 2.45 μM, 80.6 ± 5.82 μM and 125.65 ± 0.34 μM, respectively. It also inhibited NF-κB nuclear translocation by 52.93 ± 14.14% at a concentration of 31.85 μM.
... A double collimated beam with a FWHM size of 7x3 µm, at an energy of 12.7 keV (0.9763Å) at a flux of ~4 x 10 11 ph/s and an exposure time of 7.5 ms per image was used during data-collection on an Eiger2 CdTe 16M detector (Dectris, Switzerland). Diffraction data were processed using XDS [19][20][21] and AutoPROC using StarAniso 22,23 . For processing the TS datasets, the collected datasets were initially integrated using XDS and merged and scaled using the CCP4 suite program AIMLESS 24,25 . ...
We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with millisecond time-resolution. Canonical micromesh loops are mounted on an electropneumatic piston, reactions are initiated via the liquid application method (LAMA), and finally intermediate states are cryo-trapped in liquid nitrogen. We demonstrate binding of several ligands in microcrystals of three enzymes, and trapping of reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase.
... 2 Data integration and reduction was performed using XDS. 3 The structure was solved by intrinsic phasing using ShelXT 4 and refined by the full-matrix least-squares method using SHELXL through the Olex2 GUI. 5 In general, non-hydrogen atoms with occupancies of greater than 0.5 were refined anisotropically, carbon-bound hydrogen atoms were included in idealized positions and refined using a riding model. 6 The structure has a large volume of smeared electron density in the lattice corresponding to highly disordered solvents and anions which could not be successfully modelled. ...
Polycyclic aromatic hydrocarbons (PAHs) find multiple applications ranging from fabric dyes to optoelectronic materials. Hydrogenation of PAHs is often employed for their purification or derivatization. However, separation of PAHs from their hydrogenated analogues is challenging because of their similar physical properties. An example of such is the separation of 9,10-dihydroanthracene from phenanthrene/anthracene which requires fractional distillation at high temperature (∼340 °C) to obtain pure anthracene/phenanthrene in coal industry. Herein we demonstrate a new approach for this separation at room temperature using a water-soluble interlocked cage (1) as extracting agent by host-guest chemistry. The cage was obtained by self-assembly of a triimidazole donor L·HNO3 with cis-[(tmeda)Pd(NO3)2] (M) [tmeda = N,N,N',N'-tetramethylethane-1,2-diamine]. 1 has a triply interlocked structure with an inner cavity capable of selectively binding planar aromatic guests.
... Redundant 3-wavelength multi-wavelength anomalous dispersion (MAD) data were collected at beamline PXIII of the Swiss Light Source, Villigen, Switzerland (Table S1) and processed and scaled with the programs XDS and XSCALE (31). Eight consistent Se sites were identified in SHELXD (32), site refinement and phasing was done in SHARP (33). ...
Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, inorganic polyphosphate). TTMs in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here, we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Our tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane, mediated by the single C-terminal transmembrane domain, supporting earlier studies. Additionally, we reveal from crystal structures of AtTTM1 in the presence and absence of a non-hydrolyzable ATP analog a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Our structural comparison indicates a helical hairpin may facilitate movement of the TTM domain, thereby activating the kinase. Furthermore, we conducted genetic studies to show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. We demonstrate through rational design of mutations based on the 3D structure that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch. Together, our results provide insight into the structure and function of plant TTM domains.
... A dataset with a total oscillation range of 10° and 0.1° oscillations per frame was collected from each crystal under an absorbed dose of 10 megagrays. The partial datasets collected with the automated data collection system ZOO (44) were merged, integrated, and scaled using the KAMO system (45), which integrates BLEND (46), XDS, and XSCALE (47,48). Diffraction intensities were converted to structure factors using the CCP4 suite where 5% of the unique reflections were randomly selected as a test set for the calculation of free R factor (49). ...
Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.
... X-ray diffraction data were collected at 100 K using synchrotron radiation at the 23-IDD beam line (APS, Argonne, USA). X-ray diffraction data were integrated and scaled with the XDS package (250). Molecular replacement with MOLREP using the structure of SsoPox WT (PDB code 2VC5) as model was performed (251,252). ...
Quorum sensing (QS) is a communication system used by most bacteria to adapt their behavior according to population density and synchronize different actions such as virulence and biofilm formation. QS inhibition, also called quorum quenching (QQ), is a promising strategy to control bacterial infections in humans, animals or plants. Among these strategies, the use of enzymes able to interfere with the molecular signals of bacteria is particularly interesting. The thermostable enzyme SsoPox is able to degrade certain communication molecules of Gram-negative bacteria: acyl-homoserine lactones (AHL). An enzymatic engineering strategy was conducted to increase the efficiency of this lactonase to degrade a certain type of lactones: short-chain AHL, used by a large number of pathogenic bacteria. Nine screening methods were first optimized to allow the identification of potentially highly active variants. Eight mutagenesis libraries targeting residues of loop 8, known to modulate the lactonase activity of the enzyme, were then screened leading to the isolation of variants improved up to 297-fold on the targeted AHL compared to the wild type enzyme. Resolution of the three-dimensional structure of the best performing variants allowed the identification of the molecular determinants responsible for this improved activity. The best variant was then studied in vitro to assess its ability to block the QS of Serratia sp. 39006, a model bacterium using short-chain AHL. A multiple approach combining phenotypic, proteomic and metabolomic analyses allowed to demonstrate the ability of the enzyme to disrupt many behaviors of the strain such as its ability to form biofilm, to produce antibiotics or to form the gas vesicles involved in its flotation. Finally, the activity spectrum of SsoPox and several variants was studied in depth and allowed the identification of new substrates such as peptides or β-lactams. Strong modulations of promiscuous activities caused by enzyme mutations were then revealed, highlighting their potential role in the acquisition of new enzymatic functions.
... Data collection, phasing, model building and structure refinement X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) and/or Beijing Synchrotron Radiation Facility (BSRF) and were indexed and scaled using HKL2000 (28) and XDS (29). A BsPif1-AMPPNP SeMet-substituted protein dataset collected at the K edge of Se (0.9792Å) was suitable for SAD phasing using the Autosol module of Phenix (30). ...
Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conforma-tion. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conforma-tional change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Pol␦, while the isolated Pif1 has low processivity.
Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, the evolutionary molecular mechanisms how proteins acquire these loop elements and obtain new function is poorly understood. To address this question, we study glycoside hydrolase family 19 (GH19) chitinase—an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discover that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrate that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that nature applies this strategy at the molecular level to achieve a complex biological function while maintaining the original activity in the catalytic pocket, suggesting an alternative way to design new enzyme function.
RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro- and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA- and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient RNA world PUN-like motifs may have supported the correct folding of early ribozymes.
Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.
Xenorhabdins (XRDs) are produced by Xenorhabdus species and are members of the dithiopyrrolone (DTP) class of natural products that have potent antibacterial, antifungal and anticancer activity. The amide moiety of their DTP core can be methylated or not to fine-tune the bioactivity properties. However, the enzyme responsible for the amide N-methylation remained elusive. Here, we identified and characterized the amide methyltransferase XrdM that is encoded nearly 600 kb away from the XRD gene cluster using proteomic analysis, methyltransferase candidate screening, gene deletion, and allied approaches. In addition, crystallographic analysis and site-directed mutagenesis proved that XrdM is completely distinct from the recently reported DTP methyltransferase DtpM, and that both have been tailored in a species-specific manner for DTP biosynthesis in Gram-negative/positive organisms. Our study expands the limited knowledge of post-NRPS amide methylation in DTP biosynthesis and reveals the evolution of two structurally completely different enzymes for the same reaction in different organisms.
Defining chemical processes with equations is the first important step in characterizing equilibria for the assembly of supramolecular complexes, and the stoichiometry of the assembled components must be defined to generate the equation. Recently, this subject has attracted renewed interest, and statistical and/or information-theoretic measures were introduced to examine the validities of the equilibrium models used during curve fitting analyses of titration. The present study shows that these measures may not always be appropriate for credibility examinations and that further reformation of the protocols used to determine the overall stoichiometry is necessary. Hydrocarbon cage hosts and their chloroform complexes formed via weak CH-π hydrogen bonds were studied, which allowed us to introduce van ’t Hoff analyses for effective validation of the stoichiometries of supramolecular complexes. This study shows that the stoichiometries of supramolecular complexes should be carefully examined by adopting multiple measures with different origins.
Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that Nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity one in the catalytic pocket, suggesting an alternative way to design new enzyme function.
Noroviruses are the leading cause of outbreaks of acute gastroenteritis. These viruses usually interact with histo-blood group antigens (HBGAs), which are considered essential cofactors for norovirus infection. This study structurally characterizes nanobodies developed against the clinically important GII.4 and GII.17 noroviruses with a focus on the identification of novel nanobodies that efficiently block the HBGA binding site. Using X-ray crystallography, we have characterized nine different nanobodies that bound to the top, side, or bottom of the P domain. The eight nanobodies that bound to the top or side of the P domain were mainly genotype specific, while one nanobody that bound to the bottom cross-reacted against several genotypes and showed HBGA blocking potential. The four nanobodies that bound to the top of the P domain also inhibited HBGA binding, and structural analysis revealed that these nanobodies interacted with several GII.4 and GII.17 P domain residues that commonly engaged HBGAs. Moreover, these nanobody complementarity-determining regions (CDRs) extended completely into the cofactor pockets and would likely impede HBGA engagement. The atomic level information for these nanobodies and their corresponding binding sites provide a valuable template for the discovery of additional "designer" nanobodies. These next-generation nanobodies would be designed to target other important genotypes and variants, while maintaining cofactor interference. Finally, our results clearly demonstrate for the first time that nanobodies directly targeting the HBGA binding site can function as potent norovirus inhibitors. IMPORTANCE Human noroviruses are highly contagious and a major problem in closed institutions, such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics. We successfully developed and characterized four norovirus nanobodies that bound at the HBGA pockets. Compared with previously developed norovirus nanobodies that inhibited HBGA through disrupted particle stability, these four novel nanobodies directly inhibited HBGA engagement and interacted with HBGA binding residues. Importantly, these new nanobodies specifically target two genotypes that have caused the majority of outbreaks worldwide and consequently would have an enormous benefit if they could be further developed as norovirus therapeutics. To date, we have structurally characterized 16 different GII nanobody complexes, a number of which block HBGA binding. These structural data could be used to design multivalent nanobody constructs with improved inhibition properties.
Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that Nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity one in the catalytic pocket, suggesting an alternative way to design new enzyme function.
The tetrapyralzolylpyridyl diborate pentadentate ligand B2Pz4Py has been complexed to Ti(III) to form the chloro complex (B2Pz4Py)Ti(III)Cl, which is a convenient starting material for preparing alkyl and hydride derivatives of this ligand. The former (R = CH3 and CH2SiMe3) are highly thermally stable and do not react with dihydrogen to form (B2Pz4Py)Ti(III)H. Rather, treatment of the chloro starting material with NaHBEt3 affords the desired hydrido complex in 85% yield. This Ti(III) hydride was fully characterized and exists in both solution and the solid state as a dimeric species; dissociation into monomers faces a high barrier of over 40 kcal/mol, according to Density Function Theory computations. This is due to stabilization of the dimer by dispersion forces. The computations show that the dimer has an S = 1 ground state, but in solution, partial dissociation into an intermediate dimer which is an open shell singlet is possible, which accounts for the lower than expected magnetic susceptibility of 1.93 per dimer. Full dissociation into reactive monomers does not occur, based on the observed lack of reactivity with carbon dioxide. All of these com-pounds react with water to form a mu-oxo dinuclear species, which reacts further with dioxygen to form oxidized peroxo and oxo Ti(IV) complexes. All three of these compounds were fully characterized.
The achievement of targeted properties in spin crossover (SCO) materials is complicated by often unpredictable cooperative interactions in the solid state. Herein, we report a dinuclear Fe(II) triple helicate 1, which is a rare example of a SCO material possessing two distinct magnetic behaviors that depend upon the thermal scan rate. Desolvated 1 was seen to undergo spin transition (ST) which was complete following slow cooling (1 K min-1), but incomplete ST (corresponding to 50% conversion) on fast cooling (10 K min-1). The incomplete ST observed in the latter case was accompanied by a higher temperature onset of ST, differing from TIESST (Temperature-Induced Excited Spin-State Trapping) materials. The two SCO pathways have been shown to arise from the interconversion between two structural phases (a and b), with both phases having associated high spin (HS) and low spin (LS) states. SCXRD (Single Crystal X-ray Diffraction) experiments using controlled cooling rates and a synchrotron light source enabled short collection times (2-3 minutes per dataset) which has enabled the identification of a mechanism by which the slow-cooled material may fully relax. In contrast, fast-cooled materials exhibit disordered arrangements of multiple structural phases, which has in turn revealed that the [HS-LS] ↔ [LS-HS] equilibria are controllable in the solid by varying the scan rate. Such behavior has been previously observed in solution studies, but its control in solids has not been reported up to now. This study demonstrates how intermolecular cooperativity can allow multiple distinct magnetic behaviors, and provides some insight into how [HS-LS] ↔ [LS-HS] equilibria can be controlled in the solid state, which may assist in the design of next-generation logic and signaling devices.
The achievement of targeted properties in spin crossover (SCO) materials is complicated by often unpredictable cooperative interactions in the solid state. Herein, we report a dinuclear Fe(II) triple helicate 1, which is a rare example of a SCO material possessing two distinct magnetic behaviors that depend upon the thermal scan rate. Desolvated 1 was seen to undergo spin transition (ST) which was complete following slow cooling (1 K min-1), but incomplete ST (corresponding to 50% conversion) on fast cooling (10 K min-1). The incomplete ST observed in the latter case was accompanied by a higher temperature onset of ST, differing from TIESST (Temperature-Induced Excited Spin-State Trapping) materials. The two SCO pathways have been shown to arise from the interconversion between two structural phases (a and b), with both phases having associated high spin (HS) and low spin (LS) states. SCXRD (Single Crystal X-ray Diffraction) experiments using controlled cooling rates and a synchrotron light source enabled short collection times (2-3 minutes per dataset) which has enabled the identification of a mechanism by which the slow-cooled material may fully relax. In contrast, fast-cooled materials exhibit disordered arrangements of multiple structural phases, which has in turn revealed that the [HS-LS] ↔ [LS-HS] equilibria are controllable in the solid by varying the scan rate. Such behavior has been previously observed in solution studies, but its control in solids has not been reported up to now. This study demonstrates how intermolecular cooperativity can allow multiple distinct magnetic behaviors, and provides some insight into how [HS-LS] ↔ [LS-HS] equilibria can be controlled in the solid state, which may assist in the design of next-generation logic and signaling devices.
The assembly of multifunctional polynuclear coordination cages exhibiting spin-crossover (SCO) proves to be a challenge to researchers. Previous investigations into the magnetic properties of a large cubic metallosupramolecular cage, [Fe8Pd6L8]28+, constructed using semi-rigid metalloligands and encompassing an internal void of 41 Å3, found that the Fe(II) centres that occupied the corners of the cubic structure did not undergo a spin-transition. In this work, substitution of the linker metal on the face of the cage resulted in the onset of spin crossover, as evidenced by magnetic susceptibility, Mӧssbauer and single crystal X-ray diffraction. Structural comparisons of these two cages were undertaken to shed light on the possible mechanism responsible for switching of the [Fe8M(II)6L8]28+ architecture from SCO inactive to active by simply changing in the identity of M(II). This led to the suggestion that a possible interplay of intra- and intermolecular interactions may permit SCO in the Ni(II) analogue, 1. The distorted octahedral coordination environment of the secondary Ni(II) centres occupying the cage faces provided conformational flexibility for the eight metalloligands of the cubic architecture relative to the square planar Pd(II) environment. Meanwhile the occupation of axial coordination sites of the Ni(II) cations by CH3CN prevented the close packing of cages observed for the Pd(II) analogue, leading to a more offset, distant packing arrangement of cages in the lattice, whereby important areas of the cage that were shown to change most dramatically with SCO experienced a lesser degree of steric hindrance to conformational changes upon SCO. Design through selectivity of secondary metal centres on the flexibility of metalloligand structures and the effect of axial donors packing arrangements may serve as new routes in the engineering of SCO or non-SCO cage systems.
La N-myristoylation correspond à l’ajout d’un groupement myristate - un acide gras saturé de 14 carbones - sur une glycine N-terminale d’une protéine cible. Cette modification essentielle des Eucaryotes est catalysée en grande partie par la N-myristoyltransférase (NMT) et permet aux protéines ciblées d’être orientées vers des membranes spécifiques. Ainsi localisées, les protéines, via l’interaction avec d'autres partenaires, peuvent contribuer activement au fonctionnement de voies de signalisation indispensables à la cellule. La dérégulation de ce système est associée à de nombreuses pathologies humaines. Cependant, les cibles de la modification ne sont généralement pas identifiées dans les analyses protéomiques classiques du fait de limitations techniques inhérentes aux approches disponibles. Il en résulte une perte d’information importante dans la compréhension de mécanismes fondamentaux du fonctionnement cellulaire normal et pathologique. Dans ce contexte, l’objectif de ma thèse a été de développer un nouvel outil biotechnologique permettant d’approfondir et combler ce manque d’information concernant la myristoylation.Ainsi, j’ai cloné et purifié la protéase IpaJ de la bactérie pathogène Shigella flexneri. IpaJ est capable de retirer la glycine N-terminale myristoylée. J’ai démontré la très grande spécificité d’IpaJ pour ces deux seuls groupes grâce à un test en spectrométrie de masse de suivi d’activité que j’ai mis au point. Ma caractérisation de l’activité d’IpaJ indique qu’elle est bien adaptée à l’étude du myristoylome, l’ensemble des protéines myristoylées d’un protéome. J’ai alors introduit l’utilisation d’IpaJ dans une étape d’analyse protéomique de type « shotgun » et démontré sa pertinence pour l’étude du myristoylome. J’ai montré dans une analyse parallèle que la NMT peut réaliser aussi la myristoylation de lysines, ce qui était inconnu jusqu’alors. J’ai utilisé IpaJ pour distinguer les deux modifications lorsqu’elles sont en compétition sur la même protéine. Globalement, mon étude démontre qu’IpaJ constitue un nouvel outil de valeur pour l’étude de la myristoylation des glycines et des lysines.
N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate's reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.
This chapter deals with indexing single-crystal data obtained using monochromatic radiation. Such data contain error-affected coordinates of vectors of a (reciprocal) lattice. Formally, the indexing problem comes down to calculating a basis of the lattice from these coordinates.
BORON İN ENDUSTRY AND HEALTH
p>Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes; each lobe contributes an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. Knowledge of the protonation states of these aspartates in the tetrahedral intermediate state would determine the catalytic mechanism by which the enzyme operates. The three dimensional structure of endothiapepsin bound to the transition state analogue inhibitor H261 has been solved to high resolution (2.1A) using quasi Laue neutron diffraction. At the time of writing this is the largest protein structure determined at high resolution using neutron diffraction. The position of deuterium atoms in the active site indicates that the outer oxygen of Asp215 and the inhibitory hydroxyl group are protonated in the transition state analogue complex. The three dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80, 794, PD-129, 541 and PD-130,328) have also been solved using X-rays to atomic resolution allowing full anisotropic modelling of each complex. The structure of endothiapepsin complexed with the gem- diol based inhibitor PD-135, 040 has also been solved to a resolution of 1.6A. The active sites of the six structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80, 794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp 32. A number of short hydrogen bonds that may have a role in catalysis (~2.6 A) have been identified within the active site in each structure; the presence of these bonds has been confirmed using NMR techniques.</p
The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe–kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.
Ergothioneine (ET) is the 2-thiourea derivative of trimethylhistidine that is biosynthesized only by select fungi and bacteria, notably Mycobacterium tuberculosis, and functions as a potent scavenger of reactive oxygen species. Although ET is obtained in the diet and accumulates in vertebrate cells via an ET-specific transporter, the extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd_1642-1643 in Streptococcus pneumoniae D39, a Gram-positive respiratory pathogen, encodes a novel ergothioneine uptake ATP-binding cassette (ABC) transporter, which we designate EgtUV. EgtU is a permease-solute binding domain (SBD) fusion protein, and the SBD binds ET with high affinity and exquisite specificity in the cleft between the two subdomains, with cation-π interactions engaging the betaine moiety and a water-mediated hydrogen bonding network surrounding the C2-sulfur-containing imidazole ring. Bioinformatics studies reveal that EgtUV is uniquely strongly conserved among known quaternary amine-specific transporters and widely distributed in firmicutes, including the human pathogens Listeria monocytogenes, as BilEB, Enterococcus faecalis and Staphylococcus aureus. This discovery significantly diversifies the LMW thiol pool in Gram-positive human pathogens that may contribute to antioxidant defenses in the infected host.
Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α 1B AR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α 1B AR structure allows the identification of two unique secondary binding pockets. By structural comparison of α 1B AR with α 2 ARs, and by constructing α 1B AR-α 2C AR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α 1B AR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
- Le Page
LE PAGE, Y. (1982). J. Appl. Cryst. 15, 255-259.
- L C Bernstein
ANDREWS, L. C. & BERNSTEIN, H. J. (1988). Acta Cryst. A44,
1009-1018.