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.

Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

2004 ◽  
Vol 91 (01) ◽  
pp. 111-118 ◽  
Author(s):  
Tatsushiro Tamura ◽  
Jun Yamanouchi ◽  
Shigeru Fujita ◽  
Takaaki Hato
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Binding Site ◽  
Thrombus Formation ◽  
Direct Interaction ◽  
Binding Pocket ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Critical Residues

SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

scholarly journals Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiwen Liu ◽  
Fanglong Zhao ◽  
Boyang Zhao ◽  
Jie Yang ◽  
Joseph Ferrara ◽  
...  
Keyword(s):  
High Resolution ◽  
Organic Synthesis ◽  
Indole Alkaloids ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Computational Studies ◽  
X Ray ◽  
Evolutionary Branch ◽  
Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

scholarly journals Structural basis for the functional properties of the P2X7 receptor for extracellular ATP

2021 ◽  
Author(s):  
Lin-Hua Jiang ◽  
Emily A. Caseley ◽  
Steve P. Muench ◽  
Sébastien Roger
Keyword(s):  
Functional Properties ◽  
P2x7 Receptor ◽  
Site Directed Mutagenesis ◽  
P2x Receptor ◽  
Structural Basis ◽  
Structure Characteristic ◽  
Purinergic Signalling ◽  
Directed Mutagenesis ◽  
Important Mediator ◽  
Receptor Family

AbstractThe P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure–function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor.

scholarly journals The role of Val68(E11) in ligand binding to sperm whale myoglobin. Site-directed mutagenesis of a synthetic gene.

1990 ◽  
Vol 265 (20) ◽  
pp. 11788-11795
Author(s):  
K D Egeberg ◽  
B A Springer ◽  
S G Sligar ◽  
T E Carver ◽  
R J Rohlfs ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Sperm Whale ◽  
Site Directed Mutagenesis ◽  
Synthetic Gene ◽  
Directed Mutagenesis ◽  
Sperm Whale Myoglobin

scholarly journals Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor

2003 ◽  
Vol 371 (2) ◽  
pp. 443-449 ◽  
Author(s):  
Frank NEUSCHÄFER-RUBE ◽  
Eva ENGEMAIER ◽  
Sina KOCH ◽  
Ulrike BÖER ◽  
Gerhard P. PÜSCHEL
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Membrane Receptors ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Receptor Subtypes ◽  
Plasma Membrane Receptors

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF2α and PGD2. Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF2α-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

scholarly journals Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor

2019 ◽  
Vol 44 (5) ◽  
pp. 303-310 ◽  
Author(s):  
Jean-Baptiste Chéron ◽  
Amanda Soohoo ◽  
Yi Wang ◽  
Jérôme Golebiowski ◽  
Serge Antonczak ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Taste Receptor ◽  
Receptor Activation ◽  
Sweet Taste ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Sweet Taste Receptor

Abstract Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

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