Phenotypic classification of mutants: a tool for understanding ligand binding and activation of muscarinic acetylcholine receptors

2007 ◽  
Vol 35 (4) ◽  
pp. 742-745 ◽  
Author(s):  
E.C. Hulme ◽  
M.S. Bee ◽  
J.A. Goodwin
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Muscarinic Acetylcholine Receptors ◽  
Amino Acid Side Chain ◽  
Binding Modes ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Structural Stabilization ◽  
Activated State

GPCRs (G-protein-coupled receptors) such as the M1 muscarinic receptor have so far proved recalcitrant to direct structure determination. Nevertheless systematic mutagenesis, particularly alanine scanning, has advanced our understanding of their structure–function relationships. GPCRs exhibit multiple conformational states with different affinities for and abilities to activate their cognate G-proteins. Ligand binding alters these conformational equilibria, thus promoting or inhibiting signalling. Alanine-scanning mutagenesis probes the relative contributions of a particular amino acid side chain to the stability of the ground and activated states of the receptor and its complexes. These determine the phenotype of the mutant receptor. Classification of the phenotypes suggests functional roles for particular amino acid side chains, allowing us to group them accordingly. From a rhodopsin-based homology model of the M1 mAChR, a coherent view emerges of how these clusters of residues function in ligand anchoring, transduction of binding energy, global structural stabilization and selective stabilization of the ground state or the activated state of the receptor. We can identify differences in ligand-binding modes, and suggest inter- and intra-molecular interactions that are weakened or broken, or formed or intensified during acetylcholine-induced activation. In due course, we may be able to extend these insights to activation by unconventional agonists.

scholarly journals Using large-scale mutagenesis to guide single amino acid scanning experiments

2017 ◽  
Author(s):  
Vanessa E. Gray ◽  
Ronald J. Hause ◽  
Douglas M. Fowler
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Large Scale ◽  
Single Amino Acid ◽  
Side Chain ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Frequent Use ◽  
Single Substitution

AbstractAlanine scanning mutagenesis is a widely-used method for identifying protein positions that are important for function or ligand binding. Alanine was chosen because it is physicochemically innocuous and constitutes a deletion of the side chain at the β- carbon. Alanine is also thought to best represent the effects of other mutations; however, this assumption has not been formally tested. To determine whether alanine substitutions are always the best choice, we analyzed 34,373 mutations in fourteen proteins whose effects were measured using large-scale mutagenesis approaches. We found that several substitutions, including histidine and asparagine, are better at recapitulating the effects of other substitutions. Histidine and asparagine also correlated best with the effects of other substitutions in different structural contexts. Furthermore, we found that alanine is among the worst substitutions for detecting ligand interface positions, despite its frequent use for this purpose. Our work highlights the utility of large-scale mutagenesis data and can help to guide future single substitution mutational scans.

scholarly journals Mapping of an NH-terminal Ligand Binding Site of the Insulin Receptor by Alanine Scanning Mutagenesis

1995 ◽  
Vol 270 (7) ◽  
pp. 3012-3016 ◽  
Author(s):  
Paul F. Williams ◽  
Dennis C. Mynarcik ◽  
Gui Qin Yu ◽  
Jonathan Whittaker
Keyword(s):  
Ligand Binding ◽  
Insulin Receptor ◽  
Binding Site ◽  
Ligand Binding Site ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Terminal Ligand

scholarly journals Talin activates integrins by altering the topology of the β transmembrane domain

2012 ◽  
Vol 197 (5) ◽  
pp. 605-611 ◽  
Author(s):  
Chungho Kim ◽  
Feng Ye ◽  
Xiaohui Hu ◽  
Mark H. Ginsberg
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Outer Membrane ◽  
Transmembrane Domain ◽  
Side Chain ◽  
Amino Acid Side Chain ◽  
Integrin Activation ◽  
Integrin Αiibβ3 ◽  
Environmentally Sensitive ◽  
Extracellular Side

Talin binding to integrin β tails increases ligand binding affinity (activation). Changes in β transmembrane domain (TMD) topology that disrupt α–β TMD interactions are proposed to mediate integrin activation. In this paper, we used membrane-embedded integrin β3 TMDs bearing environmentally sensitive fluorophores at inner or outer membrane water interfaces to monitor talin-induced β3 TMD motion in model membranes. Talin binding to the β3 cytoplasmic domain increased amino acid side chain embedding at the inner and outer borders of the β3 TMD, indicating altered topology of the β3 TMD. Talin’s capacity to effect this change depended on its ability to bind to both the integrin β tail and the membrane. Introduction of a flexible hinge at the midpoint of the β3 TMD decoupled the talin-induced change in intracellular TMD topology from the extracellular side and blocked talin-induced activation of integrin αIIbβ3. Thus, we show that talin binding to the integrin β TMD alters the topology of the TMD, resulting in integrin activation.

scholarly journals Alanine Scanning Mutagenesis of a Type 1 Insulin-like Growth Factor Receptor Ligand Binding Site

2001 ◽  
Vol 276 (47) ◽  
pp. 43980-43986 ◽  
Author(s):  
Jonathan Whittaker ◽  
Andreas V. Groth ◽  
Dennis C. Mynarcik ◽  
Lene Pluzek ◽  
Vibeke L. Gadsbøll ◽  
...  
Keyword(s):  
Growth Factor ◽  
Ligand Binding ◽  
Binding Site ◽  
Growth Factor Receptor ◽  
Insulin Like Growth Factor ◽  
Ligand Binding Site ◽  
Alanine Scanning Mutagenesis ◽  
Receptor Ligand ◽  
Alanine Scanning

Molecular organization of the desmoglein-plakoglobin complex

1998 ◽  
Vol 111 (14) ◽  
pp. 1941-1949 ◽  
Author(s):  
N.A. Chitaev ◽  
A.Z. Averbakh ◽  
R.B. Troyanovsky ◽  
S.M. Troyanovsky
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gradient Centrifugation ◽  
Intercellular Junctions ◽  
Molecular Organization ◽  
Cortical Actin ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Desmosomal Cadherins ◽  
E Cadherin

Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and alpha-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with alpha-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent co-immunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1–3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with alpha-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as alpha-catenin and desmosomal cadherins.

scholarly journals Structural definition of polyspecific compensatory ligand recognition by P-glycoprotein

IUCrJ ◽  
2020 ◽  
Vol 7 (4) ◽  
pp. 663-672 ◽  
Author(s):  
Christina A. Le ◽  
Daniel S. Harvey ◽  
Stephen G. Aller
Keyword(s):  
Ligand Binding ◽  
Drug Transport ◽  
Intrinsic Property ◽  
Binding Pocket ◽  
Environmental Pollutant ◽  
Drug Binding ◽  
Binding Modes ◽  
Aromatic Side Chain ◽  
Alanine Scanning Mutagenesis ◽  
P Glycoprotein

The multidrug transporter P-glycoprotein (Pgp)/ABCB1/MDR1 plays an important role in multidrug resistance (MDR) and detoxification owing to its ability to efflux an unusually large and chemically diverse set of substrates. Previous phenylalanine-to-alanine scanning mutagenesis of Pgp revealed that nearly all mutations retained full MDR function and still permitted substrate transport. This suggests that either the loss of any single aromatic side chain did not affect the ligand-binding modes or that highly adaptive and compensatory drug recognition is an intrinsic property including ligand-binding shifts that preserve function. To explore this hypothesis, the ATPase function and crystallographic localization of five single-site mutations in which the native aromatic residue directly interacted with the environmental pollutant BDE-100, as shown in previous crystal structures, were tested. Two mutants, Y303A and Y306A, showed strong BDE-100 occupancy at the original site (site 1), but also revealed a novel site 2 located on the opposing pseudo-symmetric half of the drug-binding pocket (DBP). Surprisingly, the F724A mutant structure had no detectable binding in site 1 but exhibited a novel site shifted 11 Å from site 1. ATPase studies revealed shifts in ATPase kinetics for the five mutants, but otherwise indicated a catalytically active transporter that was inhibited by BDE-100, similar to wild-type Pgp. These results emphasize a high degree of compensatory drug recognition in Pgp that is made possible by aromatic amino-acid side chains concentrated in the DBP. Compensatory recognition forms the underpinning of polyspecific drug transport, but also highlights the challenges associated with the design of therapeutics that evade efflux altogether.

scholarly journals Alanine scanning of dinitroaniline/phosphorothioamidate site of α-tubulin in plasmodium species distributed in India

2020 ◽  
Vol 26 ◽  
pp. 293-297
Author(s):  
O. M. Demchuk ◽  
P. A. Karpov ◽  
A. V. Rayevsky ◽  
S. P. Ozheredov ◽  
S. I. Spivak ◽  
...  
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Specific Binding ◽  
Plasmodium Species ◽  
Protein Docking ◽  
Amino Acid Residues ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Protein Structure Optimization ◽  
Dynamics Method

Aim. Identification of amino acid residues participating in specific binding of dinitroaniline and phosphorothioamidate compounds with α-tubulin in Plasmodium falciparum. Methods. Protein structure modelling, protein structure optimization using molecular dynamics method, ligand-protein docking, alanine scanning mutagenesis. Results. Molecular docking of canonical compounds and alanine scanning mutagenesis, indicate two key (Arg2, Val250) and one minor (Glu3) residues involved in binding of both - dinitroaniline and phosphorothioamidate compounds. At the same time, it was revealed two minor residues (Asp251, Glu254) interacting only with some members of dinitroaniline grope. Conclusions. It was identified amino acid residues predetermining existence of joint site and similar interaction of α-tubulin with dinitroani-line and phosphorothioamidate compounds in P. falciparum. Keywords: malaria, Plasmodium, α-tubulin, molecular interaction, dinitroanilines compounds, phosphorothioamidate compounds, alanine scanning mutagenesis.

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