Structure of a backtracked state reveals conformational changes similar to the state following nucleotide incorporation in human norovirus polymerase

2014 ◽  
Vol 70 (12) ◽  
pp. 3099-3109 ◽  
Author(s):  
Dmitry Zamyatkin ◽  
Chandni Rao ◽  
Elesha Hoffarth ◽  
Gabriela Jurca ◽  
Hayeong Rho ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Covalent Bond ◽  
Crystal Form ◽  
Phosphoryl Group ◽  
Nucleoside Triphosphate ◽  
Orthorhombic Crystal ◽  
Crystal Forms ◽  
Terminal Nucleotide ◽  
Thumb Domain

The RNA-dependent RNA polymerase (RdRP) from norovirus (NV) genogroup II has previously been crystallized as an apoenzyme (APO1) in multiple crystal forms, as well as as a pre-incorporation ternary complex (PRE1) bound to Mn2+, various nucleoside triphosphates and an RNA primer-template duplex in an orthorhombic crystal form. When crystallized under near-identical conditions with a slightly different RNA primer/template duplex, however, the enzyme–RNA complex forms tetragonal crystals (anisotropic data,dmin≃ 1.9 Å) containing a complex with the primer/template bound in a backtracked state (BACK1) similar to a post-incorporation complex (POST1) in a step of the enzymatic cycle immediately following nucleotidyl transfer. The BACK1 conformation shows that the terminal nucleotide of the primer binds in a manner similar to the nucleoside triphosphate seen in the PRE1 complex, even though the terminal two phosphoryl groups in the triphosphate moiety are absent and a covalent bond is present between the α-phosphoryl group of the terminal nucleotide and the 3′-oxygen of the penultimate nucleotide residue. The two manganese ions bound at the active site coordinate to conserved Asp residues and the bridging phosphoryl group of the terminal nucleotide. Surprisingly, the conformation of the thumb domain in BACK1 resembles the open APO1 state more than the closed conformation seen in PRE1. The BACK1 complex thus reveals a hybrid state in which the active site is closed while the thumb domain is open. Comparison of the APO1, PRE1 and BACK1 structures of NV polymerase helps to reveal a more complete and complex pathway of conformational changes within a single RdRP enzyme system. These conformational changes lend insight into the mechanism of RNA translocation following nucleotidyl transfer and suggest novel approaches for the development of antiviral inhibitors.

scholarly journals High-resolution structures of inhibitor complexes of human indoleamine 2,3-dioxygenase 1 in a new crystal form

2018 ◽  
Vol 74 (11) ◽  
pp. 717-724 ◽  
Author(s):  
Shukun Luo ◽  
Ke Xu ◽  
Shaoyun Xiang ◽  
Jie Chen ◽  
Chunyun Chen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Resolution ◽  
Crystal Structures ◽  
Active Site ◽  
Crystal Form ◽  
Crystal Forms ◽  
Cellular Processes ◽  
Potential Target ◽  
Indoleamine 2,3 Dioxygenase

Human indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme with important roles in many cellular processes and is a potential target for drug discovery against cancer and other diseases. Crystal structures of IDO1 in complex with various inhibitors have been reported. Many of these crystals belong to the same crystal form and most of the reported structures have resolutions in the range 3.2–2.3 Å. Here, three new crystal forms of human IDO1 obtained by introducing a surface mutation, K116A/K117A, distant from the active site are reported. One of these crystal forms diffracted to 1.5 Å resolution and can be readily used for soaking experiments to determine high-resolution structures of IDO1 in complex with the substrate tryptophan or inhibitors that coordinate the heme. In addition, this mutant was used to produce crystals of a complex with an inhibitor that targets the apo form of the enzyme under the same conditions; the structure of this complex was determined at 1.7 Å resolution. Overall, this mutant represents a robust platform for determining the structures of inhibitor and substrate complexes of IDO1 at high resolution.

scholarly journals Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase

2014 ◽  
Vol 70 (5) ◽  
pp. 1484-1490 ◽  
Author(s):  
Sally Dempster ◽  
Stephen Harper ◽  
John E. Moses ◽  
Ingrid Dreveny
Keyword(s):  
Lactate Dehydrogenase ◽  
Conformational Changes ◽  
Active Site ◽  
Anaerobic Respiration ◽  
Crystal Form ◽  
Binary Complex ◽  
Cofactor Binding ◽  
Key Enzyme ◽  
Crystallization Conditions

Lactate dehydrogenase A (LDH-A) is a key enzyme in anaerobic respiration that is predominantly found in skeletal muscle and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH. LDH-A is overexpressed in many tumours and has therefore emerged as an attractive target for anticancer drug discovery. Crystal structures of human LDH-A in the presence of inhibitors have been described, but currently no structures of the apo or binary NADH-bound forms are available for any mammalian LDH-A. Here, the apo structure of human LDH-A was solved at a resolution of 2.1 Å in space groupP4122. The active-site loop adopts an open conformation and the packing and crystallization conditions suggest that the crystal form is suitable for soaking experiments. The soaking potential was assessed with the cofactor NADH, which yielded a ligand-bound crystal structure in the absence of any inhibitors. The structures show that NADH binding induces small conformational changes in the active-site loop and an adjacent helix. A comparison with other eukaryotic apo LDH structures reveals the conservation of intra-loop interactions. The structures provide novel insight into cofactor binding and provide the foundation for soaking experiments with fragments and inhibitors.

scholarly journals Conformational changes in Apolipoprotein N-acyltransferase (Lnt)

2018 ◽  
Author(s):  
Benjamin Wiseman ◽  
Martin Högbom
Keyword(s):  
Conformational Changes ◽  
Crystal Packing ◽  
Cell Envelope ◽  
Crystal Form ◽  
Covalent Attachment ◽  
Gram Negative Bacteria ◽  
Asymmetric Unit ◽  
Cellular Functions ◽  
Crystal Forms ◽  
Potential Mode

SUMMARYIn bacteria, lipoproteins are important components of the cell envelope and are responsible for many essential cellular functions. They are produced by the post-translational covalent attachment of lipids that occurs via a sequential 3-step process controlled by three essential integral membrane enzymes. The last step of this process, unique to Gram negative bacteria, is the N-acylation of the terminal cysteine by Apolipoprotein N-acyltransferase (Lnt) to form the final mature lipoprotein. Here we report 2 crystal forms of this enzyme. In one form the enzyme crystallized with two molecules in the asymmetric unit. In one of those molecules the thioester acyl-intermediate is observed. In the other molecule, the crystal packing suggests one potential mode of apolipoprotein docking to Lnt. In the second crystal form the enzyme crystallized with one molecule in the asymmetric unit in an apparent apo-state remarkably devoid of any bound molecules in the large open substrate entry portal. Taken together, these structures suggest that the movement of the essential W237 is triggered by substrate binding and could help direct and stabilize the interaction between Lnt and the incoming substrate apolipoprotein.Graphical Abstract

scholarly journals Multiple crystal forms of human MacroD2

2020 ◽  
Vol 76 (10) ◽  
pp. 477-482
Author(s):  
Sarah Wazir ◽  
Mirko M. Maksimainen ◽  
Lari Lehtiö
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Comparison ◽  
Crystal Forms ◽  
Space Groups ◽  
Single Domain Protein ◽  
Conformational Variations ◽  
Crystallization Conditions ◽  
Binding Groove

MacroD2 is one of the three human macrodomain proteins characterized by their protein-linked mono-ADP-ribosyl-hydrolyzing activity. MacroD2 is a single-domain protein that contains a deep ADP-ribose-binding groove. In this study, new crystallization conditions for MacroD2 were found and three crystal structures of human MacroD2 in the apo state were solved in space groups P41212, P43212 and P43, and refined at 1.75, 1.90 and 1.70 Å resolution, respectively. Structural comparison of the apo crystal structures with the previously reported crystal structure of MacroD2 in complex with ADP-ribose revealed conformational changes in the side chains of Val101, Ile189 and Phe224 induced by the binding of ADP-ribose in the active site. These conformational variations may potentially facilitate design efforts of a MacroD2 inhibitor.

scholarly journals Preliminary crystallographic analysis of the N-terminal domain of FILIA, a protein essential for embryogenesis

2010 ◽  
Vol 66 (9) ◽  
pp. 1111-1114
Author(s):  
Juke Wang ◽  
Tong-Cun Zhang ◽  
Xinqi Liu
Keyword(s):  
Diffraction Data ◽  
Early Stage ◽  
Crystal Form ◽  
Crystallographic Analysis ◽  
Orthorhombic Crystal ◽  
Crystal Forms ◽  
Terminal Domain ◽  
Peg 4000 ◽  
Tetragonal Crystals ◽  
Crystallization Conditions

FILIA is a component of the subcortical maternal complex that is essential for early stage embryogenesis. Its 6×His-tagged N-terminal domain was expressed inEscherichia coliand purified to homogeneity. Two types of crystals formed under different crystallization conditions during screening. Orthorhombic crystals appeared in a solution containing 1.4 Mammonium sulfate, 0.1 MTris pH 8.2 and 12% glycerol, while tetragonal crystals were obtained using 15% PEG 4000 mixed with 0.1 MHEPES pH 7.5 and 15% 2-propanol. High-quality diffraction data were collected from the two crystal forms to resolutions of 1.8 and 2.2 Å, respectively, using synchrotron radiation. The Matthews coefficients indicated that theP212121andP41212 crystals contained two molecules and one molecule per asymmetric unit, respectively. A selenomethionine-substituted sample failed to crystallize under the native conditions, but another orthorhombic crystal form was obtained under different conditions and anomalous diffraction data were collected.

scholarly journals Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6

2014 ◽  
Vol 70 (3) ◽  
pp. 636-646 ◽  
Author(s):  
Hui Chen ◽  
Sourajit M. Mustafi ◽  
David M. LeMaster ◽  
Zhong Li ◽  
Annie Héroux ◽  
...  
Keyword(s):  
Active Site ◽  
Conformational Transition ◽  
Binding Mode ◽  
Crystal Form ◽  
Crystal Forms ◽  
X Ray ◽  
Fk506 Binding Protein ◽  
Peptide Unit ◽  
Conformational Plasticity ◽  
The Central Nervous System

The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca2+channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution fromP21andP3121 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from theP3121 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition `80s loop' is flipped in theP21crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

The structure of dihydrodipicolinate reductase (DapB) fromMycobacterium tuberculosisin three crystal forms

2009 ◽  
Vol 66 (1) ◽  
pp. 61-72 ◽  
Author(s):  
Robert Janowski ◽  
Georgia Kefala ◽  
Manfred S. Weiss
Keyword(s):  
Conformational Change ◽  
Normal Modes ◽  
Low Frequency ◽  
Pyridine Nucleotide ◽  
Crystal Form ◽  
Orthorhombic Crystal ◽  
Monoclinic Crystal ◽  
Crystal Forms ◽  
Comparison Of The Results ◽  
Unstable Product

Dihydrodipicolinate reductase (DHDPR, DapB) is an enzyme that belongs to the L-lysine biosynthetic pathway. DHDPR reduces the α,β-unsaturated cyclic imine 2,3-dihydrodipicolinic acid to yield the compound 2,3,4,5-tetrahydrodipicolinic acid in a pyridine nucleotide-dependent reaction. The substrate of this reaction is the unstable product of the preceding enzyme dihydrodipicolinate synthase (DHDPS, DapA). Here, the structure of apo-DHDPR fromMycobacterium tuberculosisis reported in two orthorhombic crystal forms, as well as the structure of DHDPR fromM. tuberculosisin complex with NADH in a monoclinic crystal form. A comparison of the results with previously solved structures of this enzyme shows that DHDPR undergoes a major conformational change upon binding of its cofactor. This conformational change can be interpreted as one of the low-frequency normal modes of the structure.

Ligand-bound structures of 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase fromMoraxella catarrhalisreveal a water channel connecting to the active site for the second step of catalysis

2015 ◽  
Vol 71 (2) ◽  
pp. 239-255 ◽  
Author(s):  
Sonali Dhindwal ◽  
Priyanka Priyadarshini ◽  
Dipak N. Patil ◽  
Satya Tapas ◽  
Pramod Kumar ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Metal Ion ◽  
Water Channel ◽  
Second Step ◽  
Phosphoryl Transfer ◽  
Water Molecules ◽  
Crystal Forms ◽  
Tetrameric Structure ◽  
Site Water

KdsC, the third enzyme of the 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic pathway, catalyzes a substrate-specific reaction to hydrolyze 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of KDO and phosphate. KdsC is a phosphatase that belongs to the C0 subfamily of the HAD superfamily. To understand the molecular basis for the substrate specificity of this tetrameric enzyme, the crystal structures of KdsC fromMoraxella catarrhalis(Mc-KdsC) with several combinations of ligands, namely metal ion, citrate and products, were determined. Various transition states of the enzyme have been captured in these crystal forms. The ligand-free and ligand-bound crystal forms reveal that the binding of ligands does not cause any specific conformational changes in the active site. However, the electron-density maps clearly showed that the conformation of KDO as a substrate is different from the conformation adopted by KDO when it binds as a cleaved product. Furthermore, structural evidence for the existence of an intersubunit tunnel has been reported for the first time in the C0 subfamily of enzymes. A role for this tunnel in transferring water molecules from the interior of the tetrameric structure to the active-site cleft has been proposed. At the active site, water molecules are required for the formation of a water bridge that participates as a proton shuttle during the second step of the two-step phosphoryl-transfer reaction. In addition, as the KDO biosynthesis pathway is a potential antibacterial target, pharmacophore-based virtual screening was employed to identify inhibitor molecules for theMc-KdsC enzyme.

scholarly journals Conformational changes in Apolipoprotein N-acyltransferase (Lnt)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin Wiseman ◽  
Martin Högbom
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Cell Envelope ◽  
Covalent Modification ◽  
Covalent Attachment ◽  
Gram Negative Bacteria ◽  
Cellular Functions ◽  
Crystal Forms ◽  
Active Site Cysteine ◽  
Open Conformation

AbstractLipoproteins are important components of the cell envelope and are responsible for many essential cellular functions. They are produced by the post-translational covalent attachment of lipids that occurs via a sequential 3-step process controlled by three integral membrane enzymes. The last step of this process, unique to Gram-negative bacteria, is the N-acylation of the terminal cysteine by Apolipoprotein N-acyltransferase (Lnt) to form the final mature lipoprotein. Here we report 2 crystal forms of Lnt from Escherichia coli. In one form we observe a highly dynamic arm that is able to restrict access to the active site as well as a covalent modification to the active site cysteine consistent with the thioester acyl-intermediate. In the second form, the enzyme crystallized in an open conformation exposing the active site to the environment. In total we observe 3 unique Lnt molecules that when taken together suggest the movement of essential loops and residues are triggered by substrate binding that could control the interaction between Lnt and the incoming substrate apolipoprotein. The results provide a dynamic context for residues shown to be central for Lnt function and provide further insights into its mechanism.

scholarly journals 1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S643-S643
Author(s):  
Maria F Mojica ◽  
Christopher Bethel ◽  
Emilia Caselli ◽  
Magdalena A Taracila ◽  
Fabio Prati ◽  
...  
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Covalent Bond ◽  
Thiol Group ◽  
Viable Cell ◽  
Free Thiol ◽  
Transition State Analogs ◽  
Cell Counts ◽  
Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

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