scholarly journals Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5290
Author(s):  
Dorota Nemecz ◽  
Maciej Ostrowski ◽  
Marc Ravatin ◽  
Frederick Saul ◽  
Grazyna Faure
Keyword(s):  
Cystic Fibrosis ◽  
Phospholipase A2 ◽  
Three Dimensional ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Presynaptic Receptors ◽  
Dimensional Structure ◽  
Protein Targets ◽  
Structural Framework ◽  
Crotalus Durissus

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic β-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1–4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

scholarly journals Synthesis, Docking, and In Vitro Anticoagulant Activity Assay of Hybrid Derivatives of Pyrrolo[3,2,1-ij]Quinolin-2(1H)-one as New Inhibitors of Factor Xa and Factor XIa

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1889 ◽  
Author(s):  
Nadezhda Novichikhina ◽  
Ivan Ilin ◽  
Anna Tashchilova ◽  
Alexey Sulimov ◽  
Danil Kutov ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Protein Targets ◽  
Ic50 Value ◽  
Anticoagulant Drugs ◽  
Factor Xia ◽  
Derivatives Of

Coagulation factor Xa and factor XIa are proven to be convenient and crucial protein targets for treatment for thrombotic disorders and thereby their inhibitors can serve as effective anticoagulant drugs. In the present work, we focused on the structure–activity relationships of derivatives of pyrrolo[3,2,1-ij]quinolin-2(1H)-one and an evaluation of their activity against factor Xa and factor XIa. For this, docking-guided synthesis of nine compounds based on pyrrolo[3,2,1-ij]quinolin-2(1H)-one was carried out. For the synthesis of new hybrid hydropyrrolo[3,2,1-ij]quinolin-2(1H)-one derivatives, we used convenient structural modification of both the tetrahydro- and dihydroquinoline moiety by varying the substituents at the C6,8,9 positions. In vitro testing revealed that four derivatives were able to inhibit both coagulation factors and three compounds were selective factor XIa inhibitors. An IC50 value of 3.68 μM for was found for the best factor Xa inhibitor and 2 μM for the best factor XIa inhibitor.

Three-dimensional structure of the apo form of the N-terminal EGF-like module of blood coagulation factor X as determined by NMR spectroscopy and simulated folding

Biochemistry ◽  
1992 ◽  
Vol 31 (26) ◽  
pp. 5974-5983 ◽  
Author(s):  
Magnus Ullner ◽  
Maria Selander ◽  
Egon Persson ◽  
Johan Stenflo ◽  
Torbjoern Drakenberg ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Blood Coagulation ◽  
Three Dimensional ◽  
Coagulation Factor ◽  
Dimensional Structure ◽  
Factor X ◽  
Three Dimensional Structure

scholarly journals Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

2011 ◽  
Vol 138 (2) ◽  
pp. 165-178 ◽  
Author(s):  
Wuyang Wang ◽  
Yassine El Hiani ◽  
Paul Linsdell
Keyword(s):  
Cystic Fibrosis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Three Dimensional Structure ◽  
Cystic Fibrosis Transmembrane ◽  
Pore Lining ◽  
Cytoplasmic Side

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl− channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

scholarly journals Modification of the N-terminus of human factor IX by defective propeptide cleavage or acetylation results in a destabilized calcium-induced conformation: effects on phospholipid binding and activation by factor XIa

1997 ◽  
Vol 323 (3) ◽  
pp. 629-636 ◽  
Author(s):  
Emiel G. C. WOJCIK ◽  
Marieke VAN DEN BERG ◽  
Swibertus R. POORT ◽  
Rogier M. BERTINA
Keyword(s):  
Metal Ion ◽  
Three Dimensional ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Dimensional Structure ◽  
Amino Terminus ◽  
Human Factor Ix ◽  
Carboxyglutamic Acid ◽  
Factor Xia ◽  
Gla Domain

The propeptide of human coagulation factor IX (FIX) directs the γ-carboxylation of the first 12 glutamic acid residues of the mature protein into γ-carboxyglutamic acid (Gla) residues. The propeptide is normally removed before secretion of FIX into the blood. However, mutation of Arg-4 in the propeptide abolishes propeptide cleavage and results in circulating profactor IX in the blood. We studied three such genetic variants, factor IX Boxtel (Arg-4 → Trp), factor IX Bendorf (Arg-4 → Leu) and factor IX Seattle C (Arg-4 → Gln). These variant profactor IX molecules bind normally to anti-FIX:Mg(II) antibodies, which indicates that the mutations do not seriously affect γ-carboxylation. Metal ion titration of the binding of variant profactor IX to conformation-specific antibodies demonstrates that the calcium-induced conformation is destabilized in the variant molecules. Also the binding of FIX Boxtel to phospholipids and its activation by factor XIa requires a high (> 5 mM) calcium concentration. The three-dimensional structure of the Gla domain of FIX in the presence of calcium indicates that the acylation of the amino-terminus, rather than the presence of the propeptide, was responsible for the destabilization of the calcium-induced conformation. In order to confirm this, the α-amino group of Tyr1 of FIX was acetylated. This chemically modified FIX showed a similar destabilization of the calcium-induced conformation to variant profactor IX. Our data imply that the amino-terminus of FIX plays an important role in stabilizing the calcium-induced conformation of the Gla domain of FIX. This conformation is important for the binding to phospholipids as well as for the activation by factor XIa. Our results indicate that mutations in FIX that interfere with propeptide cleavage affect the function of the protein mainly by destabilizing the calcium-induced conformation.

scholarly journals Characterization of Colony Morphology Variants Isolated from Pseudomonas aeruginosa Biofilms

2005 ◽  
Vol 71 (8) ◽  
pp. 4809-4821 ◽  
Author(s):  
Mary Jo Kirisits ◽  
Lynne Prost ◽  
Melissa Starkey ◽  
Matthew R. Parsek
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Solid Medium ◽  
Three Dimensional ◽  
Solid Surfaces ◽  
Colony Morphology ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Parent

ABSTRACT In this study, we report the isolation of small, rough, strongly cohesive colony morphology variants from aging Pseudomonas aeruginosa PAO1 biofilms. Similar to many of the P. aeruginosa colony morphology variants previously described in the literature, these variants autoaggregate in liquid culture and hyperadhere to solid surfaces. They also exhibit increased hydrophobicity and reduced motility compared to the wild-type parent strain. Despite the similarities in appearance of our colony morphology variant isolates on solid medium, the isolates showed a range of responses in various phenotypic assays. These variants form biofilms with significant three-dimensional structure and more biomass than the wild-type parent. To further explore the nature of the variants, their transcriptional profiles were evaluated. The variants generally showed increased expression of the psl and pel loci, which have been previously implicated in the adherence of P. aeruginosa to solid surfaces. When a mutation in the psl locus was introduced into a colony morphology variant, the colony morphology was only partially affected, but hyperadherence and autoaggregation were lost. Finally, similar colony morphology variants were found in isolates from cystic fibrosis patients. These variants displayed many of the same characteristics as the laboratory variants, suggesting a link between laboratory and cystic fibrosis biofilms.

Identification of a region of strong discrimination in the pore of CFTR

2001 ◽  
Vol 281 (4) ◽  
pp. L852-L867 ◽  
Author(s):  
Nael A. McCarty ◽  
Zhi-Ren Zhang
Keyword(s):  
Cystic Fibrosis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Structural Determinants ◽  
Anion Selectivity ◽  
Relative Conductance ◽  
Pore Domain ◽  
Cystic Fibrosis Transmembrane

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl− channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl−and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl− and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl− and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

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