Structural evidence for asymmetric ligand binding to transthyretin

2015 ◽  
Vol 71 (8) ◽  
pp. 1582-1592 ◽  
Author(s):  
Michele Cianci ◽  
Claudia Folli ◽  
Francesco Zonta ◽  
Paola Florio ◽  
Rodolfo Berni ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Binding Assays ◽  
Experimental Conditions ◽  
Affinity Ligand ◽  
Competition Binding ◽  
Preferential Binding ◽  
Minor Site ◽  
Dynamics Simulations ◽  
Effective Drugs

Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization.

scholarly journals Peptides presenting the binding site of human CD4 for the HIV-1 envelope glycoprotein gp120

2012 ◽  
Vol 8 ◽  
pp. 1858-1866 ◽  
Author(s):  
Julia Meier ◽  
Kristin Kassler ◽  
Heinrich Sticht ◽  
Jutta Eichler
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Envelope Glycoprotein ◽  
Conformational Stability ◽  
Cellular Receptor ◽  
Binding Assays ◽  
Glycoprotein Gp120 ◽  
Dynamics Simulations ◽  
Key Residues ◽  
Hiv 1

Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide–gp120 complexes. The specificity of the peptide–gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids.

scholarly journals Facilitated Dissociation of Transcription Factors from Single DNA Binding Sites

2017 ◽  
Author(s):  
Ramsey I. Kamar ◽  
Edward J. Banigan ◽  
Aykut Erbas ◽  
Rebecca D. Giuntoli ◽  
Monica Olvera de la Cruz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Single Molecule ◽  
Binding Sites ◽  
Binding Kinetics ◽  
General Mechanism ◽  
Dissociation Kinetics ◽  
Dna Binding Sites ◽  
Standard Picture ◽  
Dynamics Simulations

ABSTRACTThe binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits TF off-rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key E. coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∽1 × 104 M-1s-1, establishing that FD of Fis occurs at the single-binding-site level, and we find that the off-rate saturates at large Fis concentrations in solution. While spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that facilitated dissociation depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF whose structure differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.SIGNIFICANCE STATEMENTTranscription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by demonstrating cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites, without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome.Author ContributionsRamsey I. Kamardesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperEdward J. Banigandesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperAykut Erbasdesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperRebecca D. Giuntolidesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperMonica Olvera de la Cruzdesigned research, performed research, wrote the paperReid C. Johnsondesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperJohn F. Markodesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

scholarly journals Toxicity, Binding, and Permeability Analyses of FourBacillus thuringiensis Cry1 δ-Endotoxins Using Brush Border Membrane Vesicles of Spodoptera exigua and Spodoptera frugiperda

1999 ◽  
Vol 65 (2) ◽  
pp. 457-464 ◽  
Author(s):  
Ke Luo ◽  
David Banks ◽  
Michael J. Adang
Keyword(s):  
Binding Site ◽  
Spodoptera Frugiperda ◽  
Brush Border ◽  
Spodoptera Exigua ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Binding Assays ◽  
Competition Binding

ABSTRACT The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua andSpodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles fromS. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.

Alpha- and beta-adrenergic and muscarinic cholinergic binding sites in the bladder and urethra of the rabbit

1983 ◽  
Vol 61 (1) ◽  
pp. 61-66 ◽  
Author(s):  
Anthony Johns
Keyword(s):  
Binding Site ◽  
Dissociation Constant ◽  
Receptor Binding ◽  
Binding Sites ◽  
Binding Assays ◽  
Muscarinic Binding Sites ◽  
Rabbit Urethra ◽  
Radioligand Receptor Binding ◽  
Muscarinic Cholinergic ◽  
Quinuclidinyl Benzilate

The affinities of a number of α and β-adrenergic binding sites and muscarinic cholinergic binding sites in rabbit urethra and bladder have been determined, using specific radioligand receptor binding assays. There was a greater density of β-binding sites than α-binding sites in the bladder, while, in the urethra, there was a greater density of α-binding sites than β-binding sites. The number of α-binding sites was fourfold greater in the urethra, whereas there were fewer β-binding sites in the urethra. There were fewer muscarinic binding sites in the urethra than in the bladder. The dissociation constant for [3H]dihydroalprenolol at the β-binding site was 6.4 nM, for [3H]dihydroergocryptine at the α-binding site was 2.11 nM, and for 3H-labelled l-quinuclidinyl benzilate at the muscarinic binding site was 0.22 nM.

scholarly journals Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

2021 ◽  
Vol 7 (34) ◽  
pp. eabh2217
Author(s):  
Robin A. Corey ◽  
Wanling Song ◽  
Anna L. Duncan ◽  
T. Bertie Ansell ◽  
Mark S. P. Sansom ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Binding Site ◽  
Binding Sites ◽  
Lipid Binding ◽  
Inner Membrane ◽  
Ample Evidence ◽  
Anionic Lipid ◽  
E Coli ◽  
Bacterial Proteins ◽  
Dynamics Simulations

Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different Escherichia coli inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.

scholarly journals Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs inPseudomonas aeruginosa

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Kayley H. Janssen ◽  
Manisha R. Diaz ◽  
Matthew Golden ◽  
Justin W. Graham ◽  
Wes Sanders ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Opportunistic Pathogen ◽  
Binding Properties ◽  
Content Type ◽  
Preferential Binding ◽  
Regulatory Rnas

ABSTRACTPseudomonas aeruginosais a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCECsrA homologs are present in many bacteria. The opportunistic pathogenPseudomonas aeruginosauses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activityin vivo. Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activityin vivoandin vitroare more stringent than those for RsmA.

scholarly journals N-terminal type I modules required for fibronectin binding to fibroblasts and to fibronectin's III1 module

1997 ◽  
Vol 323 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Jane SOTTILE ◽  
Deane F. MOSHER
Keyword(s):  
Cell Surface ◽  
Binding Site ◽  
Binding Sites ◽  
Solid Phase ◽  
Deletion Mutants ◽  
Type I ◽  
Binding Assays ◽  
Terminal Part ◽  
Solid Phase Binding ◽  
Fibronectin Fibrils

Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III1 module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III1. Proteins containing the fourth and fifth type I modules (70KΔI1-3) bound specifically to III1 in solid-phase binding assays; proteins lacking I4 and I5 did not bind. N-terminal molecules containing the fourth and fifth type I modules also bound to fibroblasts, suggesting that III1-like binding sites are present on the cell surface. However, the high-affinity binding sites on fibroblasts for fibronectin or the 70 kDa protein displayed more complex determinants, inasmuch as 70 kDa deletion mutants lacking I4 and I5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both competed only partially for binding of 125I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I4 and I5 and that interactions in addition to that of I4 and I5 with III1 are important for cell-surface-mediated fibronectin polymerization.

scholarly journals Phosphoinositides regulate the TRPV1 channel via two functionally distinct binding sites

2019 ◽  
Author(s):  
Aysenur Torun Yazici ◽  
Eleonora Gianti ◽  
Marina A. Kasimova ◽  
Vincenzo Carnevale ◽  
Tibor Rohacs
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Vanilloid ◽  
Trpv1 Channel ◽  
Distinct Site ◽  
Excised Patches ◽  
Transient Receptor ◽  
Dynamics Simulations

AbstractRegulation of the heat- and capsaicin-activated Transient Receptor Potential Vanilloid 1 (TRPV1) channel by phosphoinositides is controversial. In a recent cryoEM structure, an endogenous phosphoinositide was detected in the vanilloid binding site, and phosphoinositides were proposed to act as competitive vanilloid antagonists. This model is difficult to reconcile with phosphatidylinositol 4,5- bisphosphate [PtdIns(4,5)P2] being a well established positive regulator of TRPV1. To resolve this controversy, we propose that phosphoinositides regulate TRPV1 via two functionally distinct binding sites. Our molecular dynamics simulations show that phosphatidylinositol (PtdIns) is more stable in the vanilloid binding site, whereas a distinct site responsible for activation is preferentially occupied by PtdIns(4,5)P2. Consistently, we show that in the presence of PtdIns(4,5)P2 in excised patches PtdIns partially inhibited TRPV1 activity induced by low, but not high capsaicin concentrations. In the absence of PtdIns(4,5)P2 on the other hand, PtdIns partially stimulated TRPV1 activity presumably by binding to the activating site. Overall, our data resolve a major controversy in the regulation of TRPV1.

scholarly journals Facilitated dissociation of transcription factors from single DNA binding sites

2017 ◽  
Vol 114 (16) ◽  
pp. E3251-E3257 ◽  
Author(s):  
Ramsey I. Kamar ◽  
Edward J. Banigan ◽  
Aykut Erbas ◽  
Rebecca D. Giuntoli ◽  
Monica Olvera de la Cruz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Single Molecule ◽  
Binding Sites ◽  
General Mechanism ◽  
Dissociation Kinetics ◽  
Dna Binding Sites ◽  
Bimolecular Interactions ◽  
Dynamics Simulations ◽  
Kinetics Of

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∼1×104 M−1s−1, establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.

scholarly journals Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes.

1985 ◽  
Vol 100 (1) ◽  
pp. 292-296 ◽  
Author(s):  
N McKenna ◽  
J B Meigs ◽  
Y L Wang
Keyword(s):  
Rhodamine B ◽  
Binding Sites ◽  
Cardiac Fibroblasts ◽  
Sulfonyl Chloride ◽  
Fluorescein Isothiocyanate ◽  
Actin Binding ◽  
Experimental Conditions ◽  
Leg Muscles ◽  
Nonmuscle Cells ◽  
Preferential Binding

We have investigated whether living muscle and nonmuscle cells can discriminate between microinjected muscle and nonmuscle actins. Muscle actin purified from rabbit back and leg muscles and labeled with fluorescein isothiocyanate, and nonmuscle actin purified from lamb brain and labeled with lissamine rhodamine B sulfonyl chloride, were co-injected into chick embryonic cardiac myocytes and fibroblasts. When fluorescence images of the two actins were compared using filter sets selective for either fluorescein isothiocyanate or lissamine rhodamine B sulfonyl chloride, essentially identical patterns of distribution were detected in both muscle and nonmuscle cells. In particular, we found no structure that, at this level of resolution, shows preferential binding of muscle or nonmuscle actin. In fibroblasts, both actins are associated primarily with stress fibers and ruffles. In myocytes, both actins are localized in sarcomeres. In addition, the distribution of structures containing microinjected actins is similar to that of structure containing endogenous F-actin, as revealed by staining with fluorescent phalloidin or phallacidin. Our results suggest that, at least under these experimental conditions, actin-binding sites in muscle and nonmuscle cells do not discriminate among different forms of actins.

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