Site-directed mutagenesis provides insights into the selective binding of trityl derivatives to Plasmodium falciparum dUTPase

2011 ◽  
Vol 46 (8) ◽  
pp. 3309-3314 ◽  
Author(s):  
Eliseo Recio ◽  
Alexander Musso-Buendía ◽  
Antonio E. Vidal ◽  
Gian Filippo Ruda ◽  
Ganasan Kasinathan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Selective Binding

scholarly journals Helices on interdomain interface couple catalysis in the ATPPase domain with allostery in Plasmodium falciparum GMP synthetase

2019 ◽  
Author(s):  
Santosh Shivakumaraswamy ◽  
Nivedita Pandey ◽  
Lionel Ballut ◽  
Sébastien Violot ◽  
Nushin Aghajari ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Basis ◽  
Drug Targets ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Allosteric Activation ◽  
Purine Base ◽  
Interdomain Interactions ◽  
Hydrolysis Of ◽  
Gmp Synthetase

AbstractGMP synthetase catalyzes the substitution of the C2 oxo-group of the purine base in XMP with an amino-group generating GMP, the last step in the biosynthesis of GMP. This reaction involves a series of catalytic events that include hydrolysis of Gln generating ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. Inherent to the functioning of GMP synthetases is bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain by substrates binding to the ATPPase domain, synchronization of the two catalytic events and tunnelling of ammonia from the GATase to the ATPPase domain. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and, steady-state and transient kinetic assays on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. The results map the residues critical for catalysis in the ATPPase domain to the helices α11 and α12 that are located at the interdomain interface, and the lid-loop that follows α11. This apart, perturbing interdomain interactions involving residues on α11 and α12 impairs GATase activation. These results imply that this arrangement of helices at the domain interface with residues that play roles in ATPPase catalysis as well as domain crosstalk enables coupling ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

The Plasmodium falciparum–CD36 Interaction Is Modified by a Single Amino Acid Substitution in CD36

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1814-1819 ◽  
Author(s):  
Lena Serghides ◽  
Ian Crandall ◽  
Eric Hull ◽  
Kevin C. Kain
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Binding Characteristics ◽  
Ofplasmodium Falciparum ◽  
Ph Dependent ◽  
American Society ◽  
Ph Range

CD36 is an 88-kD glycoprotein involved in the cytoadherence ofPlasmodium falciparum–parasitized erythrocytes (PE) to endothelial cells. The molecular mechanisms involved in CD36-dependent cytoadherence were examined by expressing three CD36 homologues (human, murine, and rat) in COS-7 cells and observing their PE-binding characteristics over a pH range of 6.0 to 7.4 and following iodination of these receptors. PE binding to human CD36 was pH dependent, with peak binding at pH 6.8 to 7.0, and binding was unaffected by iodination. In contrast, PE adherence to murine and rat CD36 was insensitive to changes in pH, and iodination significantly reduced binding. We further show that the differences observed in the binding phenotype of human and rodent CD36 can be attributed to a single residue. Site-directed mutagenesis of the histidine at position 242 of human CD36 to tyrosine (found in rodent CD36) conferred the binding phenotype of rodent CD36 onto human CD36. Furthermore, substitution of the tyrosine at position 242 of rat CD36 for histidine conferred the binding phenotype of human CD36 onto rat CD36. These findings suggest that residue 242 is part of, or important to the conformation of, the PE-binding domain of CD36. © 1998 by The American Society of Hematology.

The Plasmodium falciparum–CD36 Interaction Is Modified by a Single Amino Acid Substitution in CD36

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1814-1819 ◽  
Author(s):  
Lena Serghides ◽  
Ian Crandall ◽  
Eric Hull ◽  
Kevin C. Kain
Keyword(s):  
Plasmodium Falciparum ◽  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Binding Characteristics ◽  
Ofplasmodium Falciparum ◽  
Ph Dependent ◽  
American Society ◽  
Ph Range

Abstract CD36 is an 88-kD glycoprotein involved in the cytoadherence ofPlasmodium falciparum–parasitized erythrocytes (PE) to endothelial cells. The molecular mechanisms involved in CD36-dependent cytoadherence were examined by expressing three CD36 homologues (human, murine, and rat) in COS-7 cells and observing their PE-binding characteristics over a pH range of 6.0 to 7.4 and following iodination of these receptors. PE binding to human CD36 was pH dependent, with peak binding at pH 6.8 to 7.0, and binding was unaffected by iodination. In contrast, PE adherence to murine and rat CD36 was insensitive to changes in pH, and iodination significantly reduced binding. We further show that the differences observed in the binding phenotype of human and rodent CD36 can be attributed to a single residue. Site-directed mutagenesis of the histidine at position 242 of human CD36 to tyrosine (found in rodent CD36) conferred the binding phenotype of rodent CD36 onto human CD36. Furthermore, substitution of the tyrosine at position 242 of rat CD36 for histidine conferred the binding phenotype of human CD36 onto rat CD36. These findings suggest that residue 242 is part of, or important to the conformation of, the PE-binding domain of CD36. © 1998 by The American Society of Hematology.

scholarly journals Selective binding of N-acetylglucosamine to the chicken hepatic lectin

1997 ◽  
Vol 324 (2) ◽  
pp. 673-680 ◽  
Author(s):  
Louise BURROWS ◽  
Susanne T. IOBST ◽  
Kurt DRICKAMER
Keyword(s):  
Expression System ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbohydrate Recognition Domain ◽  
Selective Binding ◽  
Mannose Binding Protein ◽  
Mannose Binding ◽  
Form Part ◽  
Acetyl Group

Among Ca2+-dependent (C-type) animal lectins, the chicken hepatic lectin (CHL) is unique in displaying almost complete selectivity for N-acetylglucosamine over other monosaccharide ligands. The crystal structures of the carbohydrate-recognition domain (CRD) from serum mannose-binding protein (MBP) and of a complex between the CRD from liver MBP and the methyl glycoside of N-acetylglucosamine were used to model the binding site in CHL. Substitution of portions of CHL into the MBP framework did not substantially increase selectivity. A bacterial expression system for the CRD of CHL was developed so that specific residues predicted to be near the 2-acetamido substituent of N-acetylglucosamine could be altered by site-directed mutagenesis. The results indicate that the ligand is bound to CHL in the same orientation as it binds to liver MBP. A tyrosine and a valine residue that probably contact the the N-acetyl group have been identified. These results, together with studies of ligand-binding selectivity, suggest that these residues form part of a binding pocket for the N-acetyl group, which confers selective binding of N-acetylglucosamine.

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

2013 ◽  
Vol 37 (3) ◽  
pp. 330
Author(s):  
Yanan WANG ◽  
Xudong LIU ◽  
Linlin MU ◽  
Zhipeng LIU ◽  
Chunmei LI ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Cynoglossus Semilaevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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