scholarly journals Crystal structures of glutathione- and inhibitor-bound human GGT1: Critical interactions within the cysteinylglycine binding site

2020 ◽  
pp. jbc.RA120.016265
Author(s):  
Simon S. Terzyan ◽  
Luong T. Nguyen ◽  
Anthony W.G. Burgett ◽  
Annie Heroux ◽  
Clyde A Smith ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Crystal Structures ◽  
Active Site ◽  
Molecular Level ◽  
Amino Acid Residues ◽  
Active Site Cleft ◽  
Substrate Cleavage ◽  
Glutamyl Transpeptidase ◽  
Specific Inhibitors

Overexpression of γ-glutamyl transpeptidase(GGT1) has been implicated in an array of humandiseases including asthma, reperfusion injury,and cancer. Inhibitors are needed for therapy, butdevelopment of potent, specific inhibitors ofGGT1 has been hampered by a lack of structuralinformation regarding substrate binding andcleavage. To enhance our understanding of themolecular mechanism of substrate cleavage, wehave solved the crystal structures of humanGGT1 (hGGT1) with glutathione (a substrate)and a phosphate-glutathione analog (anirreversible inhibitor) bound in the active site.These are the first structures of any eukaryoticGGT with the cysteinylglycine region of thesubstrate-binding site occupied. These structuresand the structure of apo-hGGT reveal movementof amino acid residues within the active site as thesubstrate binds. Asn-401 and Thr-381 each formhydrogen bonds with two atoms of GSH spanningthe γ-glutamyl bond. Three different atoms ofhGGT1 interact with the carboxyl-oxygen of thecysteine of GSH. Interactions between theenzyme and substrate change as the substratemoves deeper into the active site cleft. Thesubstrate reorients and a new hydrogen bond isformed between the substrate and the oxyanionhole. Thr-381 is locked into a singleconformation as an acyl bond forms between thesubstrate and the enzyme. These data provideinsight on a molecular level into the substratespecificity of hGGT1 and provide an explanationfor seemingly disparate observations regardingthe enzymatic activity of hGGT1 mutants. Thisknowledge will aid in the design of clinicallyuseful hGGT1 inhibitors.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

Structural studies of ligands for the glycine binding site of the excitatory amino acid receptor complex: 2. 6-bromo-, 6-methyl-, and 6-styryl-1,8-ethano-4-hydro-2,3-quinoxalinedione

1994 ◽  
Vol 72 (10) ◽  
pp. 2028-2036 ◽  
Author(s):  
Maciej Kubicki ◽  
Teresa W. Kindopp ◽  
Mario V. Capparelli ◽  
Penelope W. Codding
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Crystal Structures ◽  
Excitatory Amino Acid ◽  
Glycine Receptor ◽  
Three Dimensional ◽  
Unit Cell ◽  
Geometric Constraints ◽  
Cell Parameters ◽  
Π Stacking

The crystal structures of three tricyclic quinoxalinedione derivatives, 6-bromo-1,8-ethano-4-hydro-2,3-quinoxalinedione (1), 6-methyl-1,8-ethano-4-hydro-2,3-quinoxalinedione hydrate (2), and 6-styryl-1,8-ethano-4-hydro-2,3-quinoxalinedione (3), are reported. For 1 and 2, the space groups are P21/n with the unit cell parameters for 1: a = 7.4003(5) Å, b = 8.5799(5) Å, c = 14.3127(9) Å, β = 90.639(6)°, and for 2: a = 7.0590(2) Å, b = 10.7483(3) Å, c = 13.9509(7) Å, β = 103.290(3)°. For 3, the space group is P21/c, with a = 19.3683(10) Å, b = 8.0962(16) Å, c = 19.5801(16) Å, β = 114.028(6)°. Compound 3 crystallizes with two molecules in the asymmetric part of the unit cell; in one of them the styryl group is disordered. The geometries of the 1,8-ethano-4-hydro-2,3-quinoxalinedione fragments are similar in all observations, with the differences mainly caused by the different nature of the substituents in the 6-position. Hydrogen bonds connect the molecules into three-dimensional networks. Head-to-tail π-stacking between molecules connected by a center of symmetry determines the packing modes in 1 and 2 but there is no π-stacking in the crystal structure of 3. The crystal structures of the three quinoxaline derivative ligands for the glycine receptor suggest a mode of recognition that involves an [Formula: see text]receptor hydrogen bond, a three-centre hydrogen bond to the neighboring carbonyl groups on the ligand, and π-stacking between ligand and receptor. This mode is consistent with the geometric constraints of the current binding site model but places greater emphasis on hydrogen-bond interactions.

Crystal structures of substrate-bound chitinase from the psychrophilic bacteriumMoritella marinaand its structure in solution

2014 ◽  
Vol 70 (3) ◽  
pp. 676-684 ◽  
Author(s):  
Piotr H. Malecki ◽  
Constantinos E. Vorgias ◽  
Maxim V. Petoukhov ◽  
Dmitri I. Svergun ◽  
Wojciech Rypniewski
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Domain Structure ◽  
Active Site ◽  
Substrate Binding ◽  
Glycoside Hydrolases ◽  
Substrate Binding Site ◽  
Domain Structures ◽  
Structure In Solution

The four-domain structure of chitinase 60 fromMoritella marina(MmChi60) is outstanding in its complexity. Many glycoside hydrolases, such as chitinases and cellulases, have multi-domain structures, but only a few have been solved. The flexibility of the hinge regions between the domains apparently makes these proteins difficult to crystallize. The analysis of an active-site mutant ofMmChi60 in an unliganded form and in complex with the substrates NAG4and NAG5revealed significant differences in the substrate-binding site compared with the previously determined complexes of most studied chitinases. A SAXS experiment demonstrated that in addition to the elongated state found in the crystal, the protein can adapt other conformations in solution ranging from fully extended to compact.

Natural inhibitors of thrombin

2014 ◽  
Vol 111 (04) ◽  
pp. 583-589 ◽  
Author(s):  
James Huntington
Keyword(s):  
Blood Flow ◽  
Crystal Structures ◽  
Active Site ◽  
Anion Binding ◽  
Thrombin Inhibitors ◽  
Multiple Functions ◽  
Endogenous Inhibitors ◽  
Active Site Cleft ◽  
Single Protein ◽  
Natural Inhibitors

SummaryThe serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

scholarly journals High-resolution crystal structures of the solubilized domain of porcine cytochromeb5

2015 ◽  
Vol 71 (7) ◽  
pp. 1572-1581 ◽  
Author(s):  
Yu Hirano ◽  
Shigenobu Kimura ◽  
Taro Tamada
Keyword(s):  
Hydrogen Bond ◽  
Electron Transfer ◽  
High Resolution ◽  
Crystal Structures ◽  
Axial Ligand ◽  
Amino Acid Residues ◽  
Hydrogen Bond Network ◽  
Porcine Liver ◽  
Crystal Forms ◽  
Bond Network

Mammalian microsomal cytochromeb5has multiple electron-transfer partners that function in various electron-transfer reactions. Four crystal structures of the solubilized haem-binding domain of cytochromeb5from porcine liver were determined at sub-angstrom resolution (0.76–0.95 Å) in two crystal forms for both the oxidized and reduced states. The high-resolution structures clearly displayed the electron density of H atoms in some amino-acid residues. Unrestrained refinement of bond lengths revealed that the protonation states of the haem propionate group may be involved in regulation of the haem redox properties. The haem Fe coordination geometry did not show significant differences between the oxidized and reduced structures. However, structural differences between the oxidized and reduced states were observed in the hydrogen-bond network around the axial ligand His68. The hydrogen-bond network could be involved in regulating the redox states of the haem group.

Characterization of the Interaction between Thrombin and a Pentapeptide That Interferes with Anion Binding Exosite I of the Enzyme.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1719-1719
Author(s):  
Tivadar Orban ◽  
Adrian Grozav ◽  
Michael Buckys ◽  
Valentin Gogonea3 ◽  
Michael Kalafatis4
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Binding Site ◽  
Computational Methods ◽  
Active Site ◽  
Trypsin Digestion ◽  
Anion Binding ◽  
Factor V ◽  
Amino Acid Residues ◽  
Coomassie Blue

Abstract We have recently characterized a pentapeptide, DYDYQ, from coagulation factor V (Beck et. al. 2004J. Biol. Chem.279, 3084) that inhibits both factor V activation and prothrombinase function. The pentapeptide does not interfere with the active site of thrombin but rather interferes with substrate attachment. Our aim was to characterize at the molecular level the binding site of DYDYQ on thrombin. First we used computational methods (blind and focused docking) to propose a hypothetical binding site. Blind docking of the pentapeptide (structure obtained from a 20 ns molecular dynamics simulation) on thrombin was performed using a docking grid with large spacing. This approach provided us a favorable site (−4.8 kcal/mol) that was further investigated using a smaller spaced docking grid. Hydrogen bonding was analyzed between thrombin and DYDYQ. The final free energy of binding was −9.69 kcal/mol. Amino acids Y76R77I79I82 from thrombin anion binding exosite I (ABE-I), were identified to participate in the interaction of the enzyme with DYDYQ. We next investigated the Thrombin-DYDYQ interaction following cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) and mass spectrometry. In these experiments purified thrombin was inhibited in the active site with a chloromethyl ketone, and treated with the DYDYQ peptide in the presence of EDC. Two bands were observed, one corresponding to thrombin cross linked to the peptide (CT) and another band corresponding to free thrombin (T). The proteins were either (i) stained with Coomassie blue for further digestion with trypsin or (ii) transferred to nitrocellulose membranes following by staining with Coomassie blue for treatment with cyanogen bromide (CNBr). Stained bands were isolated from the gel and subjected to trypsin digestion and liquid chromatography/mass spectrometry (LC-MS). Following trypsin digestion thrombin presence in both, T and CT samples was confirmed and the peptides identified in both samples covered approximately 63% of the entire thrombin sequence. The only difference observed between the sets of peptides obtained from T and CT following digestion with trypsin, was the peptide N78IEKISM*LEK87 (M* = oxidized Methionine), which was present in the T sample but was absent in the CT sample. These results suggest that the binding site of DYDYQ to thrombin is localized in the area of the above mentioned peptide protecting it from hydrolysis by trypsin. We next analyzed T and CT by LC-MS following CNBr digestion. Three important bands (peptide products from CNBr digestion) were detected in the sample containing the T, having an approximate molecular weight of ~4,500, ~7,500, and ~9,000. CNBr digestion products of CT lacked the median band (peptide mass: ~7,500). This band corresponded to the sequence L33…Y76ERN78IEKISM84 - as confirmed by ESI-ion trap mass spectrometry amino acid sequence of its first eleven amino acid residues. The difference between the two in gel CNBr digest profiles of T and CT, confirms the conclusion drawn from the MS analysis of the triptic digests which in turn was predicted by our computational analyses. Overall our data demonstrate that 1) amino acid residues Y76R77I79I82 from thrombin provide an interactive site for DYDYQ, and 2) our results from computational methods that identify protein-peptide interaction are valid and can be confirmed by mass spectrometry.

scholarly journals Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5′-end of the RNA substrate

2020 ◽  
Vol 295 (27) ◽  
pp. 9076-9086
Author(s):  
Yuko Takagi ◽  
Naoyuki Kuwabara ◽  
Truong Tat Dang ◽  
Koji Furukawa ◽  
C. Kiong Ho
Keyword(s):  
Trypanosoma Cruzi ◽  
Binding Site ◽  
Crystal Structures ◽  
Active Site ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Active Form ◽  
Valuable Insight ◽  
Rna Triphosphatase ◽  
Rna Triphosphatases

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20–2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5′-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

scholarly journals Structures of theMycobacterium tuberculosisGlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

2018 ◽  
Vol 74 (4) ◽  
pp. 321-331
Author(s):  
Nina M. Wolf ◽  
Hiten J. Gutka ◽  
Farahnaz Movahedzadeh ◽  
Celerino Abad-Zapatero
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Adenosine Monophosphate ◽  
Class Ii ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Oligomeric State ◽  
Fructose 1,6 Bisphosphatase ◽  
Lead Inhibitors

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) fromMycobacterium tuberculosisat 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase fromSynechocystis(strain 6803) as well as the equivalent enzyme fromThermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII fromEscherichia coliand is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII fromM. tuberculosis(MtFBPaseII) is conserved and is analogous to that described previously for theE. colienzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures ofMtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for theSynechocystisenzyme. The structural and functional insights derived from the structure ofMtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

scholarly journals Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from Methanosarcina thermophila

2006 ◽  
Vol 188 (3) ◽  
pp. 1143-1154 ◽  
Author(s):  
Sarah H. Lawrence ◽  
Kelvin B. Luther ◽  
Hermann Schindelin ◽  
James G. Ferry
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Van Der Waals Interactions ◽  
Nucleophilic Attack ◽  
Titration Calorimetry ◽  
Acetyl Phosphate ◽  
Methanosarcina Thermophila ◽  
Functional Studies ◽  
Active Site Cleft

ABSTRACT Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA1) bound in the proposed active site cleft and an additional CoA (CoA2) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [KD ], 20 μM) and low-affinity (KD , 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA1 is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA2 and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser309 and Arg310, which are proximal to the reactive sulfhydryl of CoA1. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp316, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg310 binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser309.

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