scholarly journals Molecular determinants for agonist recognition and discrimination in P2X2 receptors

2019 ◽  
Vol 151 (7) ◽  
pp. 898-911 ◽  
Author(s):  
Federica Gasparri ◽  
Jesper Wengel ◽  
Thomas Grutter ◽  
Stephan A. Pless
Keyword(s):  
Drug Targets ◽  
P2x Receptors ◽  
Carbonyl Oxygen ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Ligand Recognition ◽  
Directed Mutagenesis ◽  
Backbone Carbonyl ◽  
Molecular Determinants ◽  
Atp Analogues

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.

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.

scholarly journals Site-directed Mutagenesis Identifies Residues Involved in Ligand Recognition in the Human A2aAdenosine Receptor

1995 ◽  
Vol 270 (23) ◽  
pp. 13987-13997 ◽  
Author(s):  
Jeongho Kim ◽  
Jürgen Wess ◽  
A. Michiel van Rhee ◽  
Torsten Schöneberg ◽  
Kenneth A. Jacobson
Keyword(s):  
Site Directed Mutagenesis ◽  
Ligand Recognition ◽  
Directed Mutagenesis

Identification of Critical Residues for Ligand Binding in the Integrin β3 I-domain by Site-directed Mutagenesis

2002 ◽  
Vol 87 (04) ◽  
pp. 756-762 ◽  
Author(s):  
Jun Yamanouchi ◽  
Tatsushiro Tamura ◽  
Shigeru Fujita ◽  
Takaaki Hato
Keyword(s):  
Ligand Binding ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Ligand Recognition ◽  
Directed Mutagenesis ◽  
Different Types ◽  
Β3 Integrin ◽  
Critical Residues ◽  
Α Subunits ◽  
Putative Ligand Binding

SummaryTo define the structural basis of ligand recognition by αIIb β3, we conducted site-directed mutagenesis of residues located on the top surface of the β3 I-domain that is homologous to the I-domain of several α subunits and contains a putative ligand binding site. Here we identify D158 and N215 in β3 as novel residues critical for ligand binding. Alanine substitution of D158 or N215 abolished binding of a ligand-mimetic antibody and fibrinogen to αIIb β3 induced by different types of integrin activation. CHO cells expressing recombinant αIIb β3 bearing D158A or N215A mutation did not adhere to fibrinogen. These mutations had the same effect on ligand binding to another β3 integrin, αV β3. Compared to the αI-domain structure, the βB-βC loop containing D158 in the β3 I-domain is quite different in length and sequence. These results suggest that the structure for ligand recognition is different in the βI- and αI-domains.

scholarly journals Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

2005 ◽  
Vol 71 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Chan K. Chan Kwo Chion ◽  
Sarah E. Askew ◽  
David J. Leak
Keyword(s):  
Mutational Analysis ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Styrene Oxidation ◽  
Overlapping Reading Frames ◽  
Reading Frames

ABSTRACT Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.

scholarly journals Molecular Determinants for Ligand Recognition in P2X Receptors

2016 ◽  
Vol 110 (3) ◽  
pp. 287a
Author(s):  
Federica Gasparri ◽  
Stephan A. Pless
Keyword(s):  
P2x Receptors ◽  
Ligand Recognition ◽  
Molecular Determinants

scholarly journals Identification by Site-directed Mutagenesis of Residues Involved in Ligand Recognition and Activation of the Human A3Adenosine Receptor

2002 ◽  
Vol 277 (21) ◽  
pp. 19056-19063 ◽  
Author(s):  
Zhan-Guo Gao ◽  
Aishe Chen ◽  
Dov Barak ◽  
Soo-Kyung Kim ◽  
Christa E. Müller ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Ligand Recognition ◽  
Directed Mutagenesis

scholarly journals Alteration of Leucine Aminopeptidase from Streptomyces septatus TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis

2005 ◽  
Vol 71 (11) ◽  
pp. 7229-7235 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Methyl Ester ◽  
Leucine Aminopeptidase ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Peptide Substrates ◽  
Hydrolytic Activities

ABSTRACT To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed l-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with l-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.

scholarly journals Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors

2005 ◽  
Vol 392 (1) ◽  
pp. 103-116 ◽  
Author(s):  
Baruch Karniol ◽  
Jeremiah R. Wagner ◽  
Joseph M. Walker ◽  
Richard D. Vierstra
Keyword(s):  
Red Light ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Genomic Analyses ◽  
Biochemical Studies ◽  
Lyase Activity ◽  
Acidic Environments ◽  
Micro Organisms

Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659–3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.

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