Determinants of nucleoside specificity of a macrolide phosphotransferase

2'-macrolide phosphotransferase type I [MPH(2')-I] is an antibiotic kinase that renders many macrolides, such as erythromycin, inactive by catalyzing the transfer of a phosphate group from a nucleoside triphosphate to the hydroxyl at the 2'-position of the antibiotic. MPH(2')-I is functionally and structurally analogous to the aminoglycoside kinases (APHs). However, it is distinct from most APHs in that it utilizes GTP exclusively as its phosphate donor. We will present the crystal structure of MPH(2')-I in its apo and ternary complex forms with guanosine nucleotide and different macrolide substrates. We will compare its nucleoside-binding pocket to that of the 2''-aminoglycoside phosphotransferases [APH(2'')], a subclass of aminoglycoside kinases that are capable of utilizing GTP as a phosphate donor. To further decipher the structural basis of the nucleoside specificity of MPH(2')-I, mutations of amino acid resides in the nucleoside-binding pocket have been carried out and their effects on the binding affinity of purine nucleotides were examined by isothermal titration calorimetry. Our preliminary results show that the "gatekeeper" residue plays a role in governing the nucleoside selectivity.

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Study of 2'-macrolide phosphotransferase selectivity for different substrates

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314092961 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C703-C703

Author(s):

Jonathan Blanchet ◽

Desiree Fong ◽

Albert Berghuis

Keyword(s):

Bacterial Infections ◽

Respiratory Infections ◽

Kinetic Studies ◽

Binding Pocket ◽

Nucleoside Triphosphate ◽

Type I ◽

Amino Acid Residues ◽

Wide Range ◽

The Family ◽

Phosphate Donor

Macrolides are antibiotics that have been in use since the late 1950s to treat a wide range of bacterial infections (e.g. upper respiratory infections, skin and soft–tissue infections, stomach ulcers and some venereal diseases). The structure of these antibiotics contains a lactone ring of either 14, 15, or 16 members, with a variety of sugar moieties attached. Resistance to this class of antibiotics may result from the reaction carried out by macrolide phosphotransferases [MPHs]. MPHs belong to the family of antibiotic kinases which catalyzes the transfer of a phosphate group from a nucleoside triphosphate to a specific hydroxyl on the antibiotic. However, unlike most antibiotic kinases, MPHs utilize GTP as the phosphate donor. Specifically, 2'-macrolide phosphotransferase type I [MPH(2')-I] transfers the gamma-phosphate from GTP to the 2'-hydroxyl of 14- and 15-membered ring macrolides. Crystal structure of the ternary complexes of MPH(2')-I with both 14- and 15-membered lactone macrolides have been determined. To study the basis of substrate selectivity, we have generated mutations of several amino acid residues in the macrolide-binding pocket and examined the catalytic activities of these mutants on the different classes of macrolides, including those containing a 16-membered lactone. Furthermore, we will present kinetic studies of MPH(2')-I containing mutations in the nucleoside-binding pocket in order to study the mechanism for the enzyme's preference for GTP.

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A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525783113 ◽

2016 ◽

Vol 113 (8) ◽

pp. 2068-2073 ◽

Cited By ~ 32

Author(s):

Erik W. Debler ◽

Kanishk Jain ◽

Rebeccah A. Warmack ◽

You Feng ◽

Steven G. Clarke ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Structural Basis ◽

Titration Calorimetry ◽

Type I ◽

Histone H4 ◽

Active Site Residues ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Product Specificity

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

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The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1

International Journal of Molecular Sciences ◽

10.3390/ijms21113940 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3940 ◽

Cited By ~ 1

Author(s):

Kevin M. Lewis ◽

Chelsie L. Greene ◽

Steven A. Sattler ◽

Buhyun Youn ◽

Luying Xun ◽

...

Keyword(s):

Binding Protein ◽

Metal Chelate ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Titration Calorimetry ◽

Bacterial Cells ◽

Chelate Complexes ◽

Quantum Chemical Analysis ◽

Ligand Selectivity

The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal–chelate complexes. Microorganisms can transport free EDTA, but not metal–EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure–function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal–chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.

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Structural Basis for the Inhibition of the Autophosphorylation Activity of HK853 by Luteolin

Molecules ◽

10.3390/molecules24050933 ◽

2019 ◽

Vol 24 (5) ◽

pp. 933 ◽

Cited By ~ 1

Author(s):

Yuan Zhou ◽

Liqun Huang ◽

Shixia Ji ◽

Shi Hou ◽

Liang Luo ◽

...

Keyword(s):

Adenosine Diphosphate ◽

Binding Pocket ◽

Structural Basis ◽

Inhibition Mechanism ◽

Titration Calorimetry ◽

Two Component System ◽

Histidine Kinases ◽

Variable Environments ◽

Π Stacking Interaction ◽

Novel Target

The two-component system (TCS) is a significant signal transduction system for bacteria to adapt to complicated and variable environments, and thus has recently been regarded as a novel target for developing antibacterial agents. The natural product luteolin (Lut) can inhibit the autophosphorylation activity of the typical histidine kinase (HK) HK853 from Thermotoga maritime, but the inhibition mechanism is not known. Herein, we report on the binding mechanism of a typical flavone with HK853 by using solution NMR spectroscopy, isothermal titration calorimetry (ITC), and molecular docking. We show that luteolin inhibits the activity of HK853 by occupying the binding pocket of adenosine diphosphate (ADP) through hydrogen bonds and π-π stacking interaction structurally. Our results reveal a detailed mechanism for the inhibition of flavones and observe the conformational and dynamics changes of HK. These results should provide a feasible approach for antibacterial agent design from the view of the histidine kinases.

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Structural basis for the diversity of the mechanism of nucleotide hydrolysis by the aminoglycoside-2′′-phosphotransferases

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319015079 ◽

2019 ◽

Vol 75 (12) ◽

pp. 1129-1137

Author(s):

Clyde A. Smith ◽

Marta Toth ◽

Nichole K. Stewart ◽

Lauren Maltz ◽

Sergei B. Vakulenko

Keyword(s):

Conformational Changes ◽

Structural Basis ◽

Hydroxyl Groups ◽

Nucleotide Hydrolysis ◽

Structural Differences ◽

Phosphate Donor ◽

Aminoglycoside Phosphotransferases ◽

High Level ◽

Hydrolysis Of ◽

Level Resistance

Aminoglycoside phosphotransferases (APHs) are one of three families of aminoglycoside-modifying enzymes that confer high-level resistance to the aminoglycoside antibiotics via enzymatic modification. This has now rendered many clinically important drugs almost obsolete. The APHs specifically phosphorylate hydroxyl groups on the aminoglycosides using a nucleotide triphosphate as the phosphate donor. The APH(2′′) family comprises four distinct members, isolated primarily from Enterococcus sp., which vary in their substrate specificities and also in their preference for the phosphate donor (ATP or GTP). The structure of the ternary complex of APH(2′′)-IIIa with GDP and kanamycin was solved at 1.34 Å resolution and was compared with substrate-bound structures of APH(2′′)-Ia, APH(2′′)-IIa and APH(2′′)-IVa. In contrast to the case for APH(2′′)-Ia, where it was proposed that the enzyme-mediated hydrolysis of GTP is regulated by conformational changes in its N-terminal domain upon GTP binding, APH(2′′)-IIa, APH(2′′)-IIIa and APH(2′′)-IVa show no such regulatory mechanism, primarily owing to structural differences in the N-terminal domains of these enzymes.

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Structural Insights into Azole-based Inhibitors of Heme Oxygenase-1: Development of Selective Compounds for Therapeutic Applications

Current Medicinal Chemistry ◽

10.2174/0929867325666180606082512 ◽

2019 ◽

Vol 25 (42) ◽

pp. 5803-5821 ◽

Cited By ~ 3

Author(s):

Mona N. Rahman ◽

Dragic Vukomanovic ◽

Jason Z. Vlahakis ◽

Walter A. Szarek ◽

Kanji Nakatsu ◽

...

Keyword(s):

Heme Oxygenase ◽

Competitive Inhibition ◽

Cytochromes P450 ◽

Structural Similarity ◽

Binding Pocket ◽

Initial Study ◽

Structural Basis ◽

Heme Oxygenase 1 ◽

Design Strategies ◽

Inhibitor Binding

The development of isozyme-selective heme oxygenase (HO) inhibitors promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties with a role in several disease states; thus, it is an enticing therapeutic target. Historically, the metalloporphyrins have been used as competitive HO inhibitors based on their structural similarity to the substrate, heme. However, heme’s important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), results in non-selectivity being an unfortunate side effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort over a decade ago to develop novel compounds as potent, selective inhibitors of HO. The result was the creation of the first generation of non-porphyrin based, non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated and provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. Notably, HO-1 inhibitors are of particular interest for the treatment of hyperbilirubinemia and certain types of cancer. Key features based on this initial study have already been used by others to discover additional potential HO-1 inhibitors. Moreover, studies have begun to use selected compounds and determine their effects in some disease models.

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Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization

Nature Communications ◽

10.1038/s41467-021-25248-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christopher Agnew ◽

Pelin Ayaz ◽

Risa Kashima ◽

Hanna S. Loving ◽

Prajakta Ghatpande ◽

...

Keyword(s):

Md Simulations ◽

Kinase Domain ◽

Structural Basis ◽

Type I ◽

Type Ii ◽

Exchange Mass Spectrometry ◽

Receptor Complexes ◽

Bmp Receptors ◽

Morphogenetic Protein ◽

Smad Proteins

AbstractUpon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

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Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽

10.7554/elife.22153 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 94

Author(s):

Akira Karasawa ◽

Toshimitsu Kawate

Keyword(s):

P2x7 Receptor ◽

Hydrophobic Interactions ◽

Binding Pocket ◽

Drug Binding ◽

Structural Basis ◽

Atp Binding ◽

Allosteric Site ◽

Channel Opening ◽

A Site ◽

Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

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Cryo-EM Structures of Prestin and the Molecular Basis of Outer Hair Cell Electromotility

10.1101/2021.08.06.455374 ◽

2021 ◽

Author(s):

Navid Bavi ◽

Michael D Clark ◽

Gustavo F Contreras ◽

Rong Shen ◽

Bharat Reddy ◽

...

Keyword(s):

Outer Hair Cell ◽

Binding Pocket ◽

Structural Features ◽

Outer Hair Cells ◽

Anion Binding ◽

Structural Basis ◽

Inhibition Mechanism ◽

Core Domain ◽

The Core ◽

Voltage Dependent

The voltage-dependent motor protein, Prestin (SLC26A5) is responsible for the electromotive behavior of outer hair cells (OHCs). Here, we determined the structure of dolphin Prestin in complex with Cl- and the inhibitor Salicylate using single particle cryo-electron microscopy. These structures establish the specific structural features of mammalian Prestin and reveal small but significant differences with the transporter members of the SLC26 family of membrane proteins. Comparison with SLC26A9 point to conformational differences in the special relationship between the core and gate domains. Importantly, we highlight substantial alterations to the hydrophobic footprint of Prestin as it relates to the membrane, which point to a potential influence of Prestin on its surrounding lipid. The structure of Prestin bound to the inhibitor Salicylate confirms the nature of the anion binding pocket, formed by TM3 and TM10 in the Core domain and a set of anion coordinating residues which include Q97, F101, F137, S398 and R399. The presence of a well-defined density for Salycilate points to an inhibition mechanism based on competition for the anion-binding pocket of Prestin. These observations illuminate the structural basis of Prestin electromotility, a key component in the mammalian cochlear amplifier.

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