scholarly journals Noncontiguous regions in the extracellular domain of EGF receptor define ligand-binding specificity.

1991 ◽  
Vol 2 (5) ◽  
pp. 337-345 ◽  
Author(s):  
I Lax ◽  
R Fischer ◽  
C Ng ◽  
J Segre ◽  
A Ullrich ◽  
...  
Keyword(s):  
Binding Energy ◽  
Ligand Binding ◽  
Binding Site ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Binding Properties ◽  
Amino Terminal ◽  
High Affinity ◽  
Domain Iii ◽  
Domain I

Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand-binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high-affinity binding site typical of human EGFR.

scholarly journals Domains III and I-2α, at the Entrance of the Binding Cleft, Play an Important Role in Cold Adaptation of the Periplasmic Dipeptide-Binding Protein (DppA) from the Deep-Sea Psychrophilic Bacterium Pseudoalteromonas sp. Strain SM9913

2010 ◽  
Vol 76 (13) ◽  
pp. 4354-4361 ◽  
Author(s):  
Wei-Xin Zhang ◽  
Bin-Bin Xie ◽  
Xiu-Lan Chen ◽  
Sheng Dong ◽  
Xi-Ying Zhang ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Cold Adaptation ◽  
Binding Protein ◽  
Structural Instability ◽  
Peptide Transporter ◽  
Psychrophilic Bacterium ◽  
Binding Cleft ◽  
Domain Iii ◽  
Domain I

ABSTRACT The peptide transporter from a cold-adapted bacterium has never been reported. In the present study, the dpp operon from the psychrophilic bacterium Pseudoalteromonas sp. strain SM9913 was cloned and analyzed. The dipeptide binding protein DppA of SM9913 was overexpressed in Escherichia coli, and its cold adaptation characteristics were studied. The recombinant DppA of SM9913 (PsDppA) displayed the highest ligand-binding affinity at 15°C, whereas the recombinant DppA of E. coli (EcDppA) displayed the highest ligand-binding affinity at 35°C. Thermal and guanidium hydrochloride unfolding analyses indicated that PsDppA has more structural instability than EcDppA. Six domain-exchanged mutants of PsDppA were expressed and purified. Analyses of these mutants indicated that domains III, I-2, and I-3 of PsDppA were less stable than those from EcDppA and that domains III and I-2 made a significant contribution to the high binding affinity of PsDppA at low temperatures. Structural and sequence analyses suggested that the state transition-involved regions in domain III and the α part of domain I-2 are the hot spots of optimization during cold adaptation and that decreasing the side-chain size in these regions is an important strategy for the cold adaptation of PsDppA.

Generation of an agonistic binding site for blockers of the M3 muscarinic acetylcholine receptor

2008 ◽  
Vol 412 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Doreen Thor ◽  
Angela Schulz ◽  
Thomas Hermsdorf ◽  
Torsten Schöneberg
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Binding Sites ◽  
Expression System ◽  
Muscarinic Acetylcholine Receptor ◽  
Intracellular Loop ◽  
Inverse Agonists ◽  
Yeast Expression System ◽  
High Affinity

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M3Rs {M3 mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3–10 μM), an inverse agonist on wild-type M3R. Many of the mutations sensitizing M3R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M3R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M3R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M3R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki ∼10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.

The Amino Acid Sequence in Fibrin Responsible for High Affinity Thrombin Binding

2001 ◽  
Vol 85 (03) ◽  
pp. 470-474 ◽  
Author(s):  
Kevin Siebenlist ◽  
Stephen Brennan ◽  
Trudy Holyst ◽  
Michael Mosesson ◽  
David Meh
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Site ◽  
Binding Affinity ◽  
Ionization Mass ◽  
Consensus Sequences ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Carboxy Terminal ◽  
Sulfated Peptides

SummaryHuman fibrin has a low affinity thrombin binding site in its E domain and a high affinity binding site in the carboxy-terminal region of its variant ’ chain (’408-427). Comparison of the ’ amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known to bind to thrombin exosites such as those in GPIb , the platelet thrombin receptor, thrombomodulin, and hirudin suggests no homology or consensus sequences, but Glu and Asp enrichment are common to all. Tyrosine sulfation in these sequences enhances thrombin exosite binding, but this has not been uniformly investigated. The fibrinogen ’ chain mass determined by electrospray ionization mass spectrometry, was 50,549 Da, a value 151 Da greater than predicted from its amino acid/carbohydrate sequence. Since each sulfate group increases mass by 80 Da, this indicates that both tyrosines at 418 and 422 are sulfated. A series of overlapping ’ peptides was prepared for evaluation of their inhibition of 125I-labeled PPACK-thrombin binding to fibrin. ’414-427 was as effective an inhibitor as ’408-427 and its binding affinity was dependent on all carboxy-terminal residues. Mono Tyr-sulfated peptides were prepared by substituting non-sulfatable Phe for Tyr at ’ 418 or 422. Sulfation at either Tyr residue increased binding competition compared with non-sulfated peptides, but was less effective than doubly sulfated peptides, which had 4 to 8-fold greater affinity. The reverse ’ peptide or the forward sequence with repositioned Tyr residues did not compete well for thrombin binding, indicating that the positions of charged residues are important for thrombin binding affinity

scholarly journals Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

scholarly journals Domain deletion in the extracellular portion of the EGF-receptor reduces ligand binding and impairs cell surface expression.

1990 ◽  
Vol 1 (2) ◽  
pp. 173-188 ◽  
Author(s):  
I Lax ◽  
F Bellot ◽  
A M Honegger ◽  
A Schmidt ◽  
A Ullrich ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Protein Tyrosine Kinase ◽  
Egf Receptor ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Amino Terminal ◽  
Mutant Receptors

Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R.

scholarly journals Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

2007 ◽  
Vol 406 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Lavinia Cicortas Gunnarsson ◽  
Cedric Montanier ◽  
Richard B. Tunnicliffe ◽  
Mike P. Williamson ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Hydrophobic Interactions ◽  
Molecular Engineering ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Rhodothermus Marinus ◽  
Binding Properties ◽  
Carbohydrate Binding Modules

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

scholarly journals Comparison of random and site directed mutation effects on the efficacy between lead SARS-CoV2 anti-protease drugs Indinavir and Hydroxychloroquine

2020 ◽  
Author(s):  
Jithin S. Sunny ◽  
Lilly M. Saleena ◽  
Saranya Balachandran ◽  
Solaipriya S.
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Binding Affinity ◽  
Continuous Distribution ◽  
Ligand Binding Site ◽  
Binding Region ◽  
Directed Mutation ◽  
Significant Difference ◽  
Polar Residues ◽  
Substitution Mutations

Abstract The diversification of virus can be attributed to random mutations leading to the development of drug resistance. The variations can be inherited from one generation to the other rendering the drug ineffective. However, a pharmacologically induced selection pressure can be countered by introducing drugs better adapted to work under rapid mutations. In this study we try to explore the effect of site directed and random substitution mutations simulated in the ligand binding region of SARS-CoV2 protease. Amongst six currently studied anti-protease drugs for COVID-19, Indinavir and Hydroxychloroquine were chosen for the study based on their high binding affinity scores, -6.81, and -4.81 respectively. The effect of mutations in protein-ligand binding was analysed in two steps. Initially, analysis of over 90 homologous protease and 100 SARS-CoV-2 orf1ab regions revealed un-conserved residues in the ligand binding sites. Gly170 and Thr190 were identified and interchanged with polar residues such as ARG, ASN and non-polar residues such as ALA, ILE. The resulting mutants were modelled, minimized and docked with Indinavir and Hydroxychloroquine. A higher binding affinity was observed for Indinavir; however, less variance in the binding affinity was observed for the latter. These results were consistent for random mutations as well. A Bio.seqIO based pipeline was build to simulate changes in the ligand binding site. Under the assumption that the ligand binding region has an equal probability of mutation over a given range for continuous distribution, 200 cycles of mutation was carried out in the nucleotide region corresponding to the ligand binding site. A paired t-test revealed a significant difference between the binding affinity of these mutant Indinavir and Hydroxychloroquine-protease complexes. Further, mean and variance was found to be higher for Indinavir-protease complex but Hydroxychloroquine displayed lesser variance pointing at a constant binding capability towards the mutant. Our study highlights the role of Hydroxychloroquine as a drug that can complement an evolving SARS-CoV2 main protease.

scholarly journals Aptamer-modified nanomaterials: principles and applications

2016 ◽  
Vol 18 (1-2) ◽  
Author(s):  
Katharina Urmann ◽  
Julia Modrejewski ◽  
Thomas Scheper ◽  
Johanna-G. Walter
Keyword(s):  
Chemical Synthesis ◽  
Binding Affinity ◽  
Nanostructured Materials ◽  
Nanostructured Material ◽  
Solid Support ◽  
Binding Properties ◽  
Promising Alternative ◽  
Nanostructured Surfaces ◽  
High Affinity

AbstractAptamers are promising alternative binders that can substitute antibodies in various applications. Due to the advantages of aptamers, namely their high affinity, specificity and stability, along with the benefits originating from the chemical synthesis of aptamers, they have attracted attention in various applications including their use on nanostructured material. This necessitates the immobilization of aptamers on a solid support. Since aptamer immobilization may interfere with its binding properties, the immobilization of aptamers has to be investigated and optimized. Within this review, we give general insights into the principles and factors controlling the binding affinity of immobilized aptamers. Specific features of aptamer immobilization on nanostructured surfaces and nanoparticles are highlighted and a brief overview of applications of aptamer-modified nanostructured materials is given.

scholarly journals A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics

2020 ◽  
Author(s):  
Jonas Gossen ◽  
Simone Albani ◽  
Anton Hanke ◽  
Benjamin P. Joseph ◽  
Cathrine Bergh ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Active Sites ◽  
Chemical Space ◽  
Protein Structures ◽  
Rational Drug Design ◽  
In Vitro Assays ◽  
Strong Basis ◽  
High Affinity ◽  
Compound Libraries

AbstractThe SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unfortunately, unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ~30,000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ~200 virtual screenings of compound libraries on selected protein structures, we redefine the protein’s druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.

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