scholarly journals High Affinity Tamoxifen Analogues Retain Extensive Positional Disorder when Bound to Calmodulin

2021 ◽  
Author(s):  
Lilia Milanesi ◽  
Clare R. Trevitt ◽  
Brian Whitehead ◽  
Andrea M. Hounslow ◽  
Salvador Tomas ◽  
...  
Keyword(s):  
Affinity Binding ◽  
Hydrophobic Pocket ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
Positional Disorder ◽  
High Affinity ◽  
Structure And Dynamics ◽  
Fluorescence Measurements ◽  
Close Proximity ◽  
Intermolecular Noe

Abstract. Using a combination of NMR and fluorescence measurements we have investigated the structure and dynamics of the complexes formed between calcium loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High affinity binding (Kd ~ 300 nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower affinity antagonists TFP, J-8 and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular NOE restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high affinity binding occurs without the loss of extensive positional dynamics.

scholarly journals High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin

2021 ◽  
Vol 2 (2) ◽  
pp. 629-642
Author(s):  
Lilia Milanesi ◽  
Clare R. Trevitt ◽  
Brian Whitehead ◽  
Andrea M. Hounslow ◽  
Salvador Tomas ◽  
...  
Keyword(s):  
Nuclear Overhauser Effect ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Positional Disorder ◽  
High Affinity ◽  
Structure And Dynamics ◽  
Fluorescence Measurements ◽  
Overhauser Effect ◽  
Close Proximity ◽  
Nuclear Overhauser

Abstract. Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding (Kd∼300 nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics.

Oxytocin receptors in the oviduct during the oestrous cycle of the ewe

1990 ◽  
Vol 124 (3) ◽  
pp. 353-359 ◽  
Author(s):  
V. J. Ayad ◽  
S. A. McGoff ◽  
D. C. Wathes
Keyword(s):  
Binding Sites ◽  
Luteal Phase ◽  
Dissociation Constants ◽  
Relative Concentration ◽  
Oestrous Cycle ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Oxytocin Receptors

ABSTRACT The presence of oxytocin receptors in ovine oviduct has been investigated. High-affinity binding sites for [3H]oxytocin were detected in crude membrane fractions prepared from the oviducts of ewes killed during the oestrous period. The dissociation constant calculated for these sites in competition studies was 1·7 nmol/l. Similar dissociation constants were calculated for [Arg8]-vasopressin and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4, Gly7]-oxytocin, indicating that these sites represent oxytocin receptors. At least one additional site of lower affinity and undetermined identity was present. The relative concentration of oxytocin-binding sites in preparations of oviduct membranes were estimated in ewes killed at different stages of the oestrous cycle using a single concentration of [3H]oxytocin. Binding was low during the luteal phase of the cycle but increased to a maximum at oestrus (77·7 fmol/mg protein). Binding fell after ovulation, reaching what appeared to be basal concentrations by the early luteal stage of the cycle. Binding to oviductal membranes from prepubertal, anoestrous and pregnant ewes was also low, but in anoestrous animals which had been treated with progesterone and oestrogen it was similar to values measured in ewes at oestrus. These results are consistent with the existence of oviductal oxytocin receptors which are regulated by ovarian steroids. We conclude that oxytocin receptors are present in the oviduct of the ewe around the time of ovulation. The significance of oxytocin to events taking place in the oviduct at this time remains to be determined. Journal of Endocrinology (1990) 124, 353–359

scholarly journals Alternative ligands as probes for the carotenoid-binding site of lobster carapace crustacyanin

1990 ◽  
Vol 265 (3) ◽  
pp. 919-921 ◽  
Author(s):  
J B Clarke ◽  
E E Eliopoulos ◽  
J B C Findlay ◽  
P F Zagalsky
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Binding Sites ◽  
Amino Acid Sequences ◽  
Fluorescence Probes ◽  
Affinity Binding ◽  
Hydrophobic Pocket ◽  
High Affinity Binding ◽  
High Affinity ◽  
Parinaric Acid

The apoproteins of the lobster carotenoprotein, crustacyanin, show single high-affinity binding sites for the hydrophobic fluorescence probes 8-anilo-1-naphthalenesulphonic acid and cis-parinaric acid, and exhibit fluorescence transfer from tryptophan to the ligands. These results, together with information from the amino acid sequences, infer that the native carotenoid, astaxanthin, is bound to each apoprotein within an internal hydrophobic pocket, or calyx.

Conformational Activation of Zymogen-Like Thrombin Variants by Tight Binding Ligands

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3070-3070
Author(s):  
Anuja Khan ◽  
Peter Panizzi ◽  
Heather K Kroh ◽  
Paul E. Bock ◽  
Sriram Krishnaswamy
Keyword(s):  
Active Site ◽  
Affinity Binding ◽  
Terminal Sequence ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fluorescence Measurements ◽  
N Terminus ◽  
Substrate Cleavage ◽  
Binding Cleft

Abstract A series of well-established structural transitions lead to proteinase formation following zymogen cleavage in the family of serine proteinases homologous to chymotrypsin. These structural changes are triggered by the formation of a salt bridge following the generation of a new N-terminus that inserts in a sequence-specific way into a binding cleft. Based on the established dependence of proteinase maturation on the N-terminal sequence, we prepared a series of recombinant variants of human thrombin (IIa) in which the I16-V17-E18 N-terminal sequence in wild type IIa (IIaWT) was replaced with I-G-E (IIaIGE), T-A-T (IIaTAT) or V-I-E (IIaVIE). In comparison to IIaWT, these variants exhibited greatly diminished ability to hydrolyze peptidyl substrates. Initial velocities measured with H-Dphenylalanyl- L-pipecolyl-L-arginine-p-nitroanilide (S2238) and L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroanilide (S2366) established a graded reduction in activity ranging from ~0.4% (IIaIGE) to < 0.2% (IIaVIE and IIaTAT) that was also dependent on the concentration of the peptidyl substrate relative to Km. These findings are expected for variants that remain zymogen-like because of an impaired ability to drive proteinase formation. This interpretation was confirmed with studies using a fragment of staphylocoagulase (SC1–325). Structural studies have established SC1–325 to function as a conformational activator of the zymogen, prethrombin 2, that is accomplished by high affinity binding and the insertion of its N-terminus into the binding cleft. Saturating concentrations of SC1–325 had a small inhibitory effect on IIaWT but restored the rate of peptidyl substrate cleavage by IIaTAT, IIaVIE and IIaIGE to levels observed with IIaWT. While SC1–325 could bind to prothrombin and prethrombin 2 with high affinity and yield catalytic activity, a deletion mutant of SC lacking 13 residues (SC13–325) produced no detectable activity in the zymogen species. This result is consistent with the essential role played by the N-terminus of SC in conformational activation. SC13–325 was found to bind to the series of IIa variants with inferred equilibrium dissociation constants of ~10−10M. Surprisingly, SC13–325 restored peptidyl substrate cleavage by the zymogen-like variants of IIa to levels seen with IIaWT. It follows that rescue of the zymogen-like variants of IIa does not require N-terminal insertion by SC. These inferences with peptidyl substrates were further pursued with binding studies examining the ability of Nα-dansyl-(p-guanidino)-L-phenylalanine-piperidide (I-2581) to bind to the active site. Fluorescence measurements indicated that I-2581 bound with high affinity to IIaWT (Kd ~5 nM) but in an undetectably weak manner to IIaIGE. However, saturating concentrations of SC13–325 could completely restore high affinity binding of I-2581 to IIaIGE. Thus, rescue of peptidyl substrate cleavage by SC13–325 is matched by a restored ability of the zymogenlike variants to accommodate ligands at the active site. The findings raise the possibility that rescue of these variants by SC arises from ligand-dependent effects that stabilize proteinase structure and overcome the sub-optimal ability of I-G-E, T-A-T or V-I-E to insert properly into the binding cleft. This possibility was further investigated using the soluble extracellular domain of thrombomodulin (sTM) which represents one of the tightest binding natural ligands for IIa. As with SC13–325, high concentrations of sTM were also found to enhance peptidyl substrate cleavage by IIaIGE. Extent of rescue of IIaIGE by sTM was also dependent on the substrate and the concentration of substrate relative to Km. In complex with sTM, IIaIGE exhibited a partial restoration in protein C activation. While we have employed these N-terminal insertion variants as prototypic zymogen-like forms of IIa, rescue of these variants by strong ligands points to a generalized allosteric mechanism by which thrombin function may be regulated. Comparable or related phenomena may underlie the regulated action of thrombin on its range of biological substrates.

scholarly journals Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2767-2773 ◽  
Author(s):  
H Shen ◽  
M Clauss ◽  
J Ryan ◽  
AM Schmidt ◽  
P Tijburg ◽  
...  
Keyword(s):  
Binding Sites ◽  
Mononuclear Phagocytes ◽  
Affinity Binding ◽  
Vascular Endothelial ◽  
Vascular Permeability Factor ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Transduction Mechanisms ◽  
Endothelial Growth Factor

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I- VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.

scholarly journals Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2767-2773 ◽  
Author(s):  
H Shen ◽  
M Clauss ◽  
J Ryan ◽  
AM Schmidt ◽  
P Tijburg ◽  
...  
Keyword(s):  
Binding Sites ◽  
Mononuclear Phagocytes ◽  
Affinity Binding ◽  
Vascular Endothelial ◽  
Vascular Permeability Factor ◽  
High Affinity Binding ◽  
Lower Affinity ◽  
High Affinity ◽  
Transduction Mechanisms ◽  
Endothelial Growth Factor

Abstract Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I- VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

Sign in / Sign up

Export Citation Format

Share Document