scholarly journals Revisiting the interaction of heme with hemopexin: Recommendations for the responsible use of an emerging drug

2020 ◽  
Author(s):  
Milena S. Detzel ◽  
Benjamin F. Syllwasschy ◽  
Francèl Steinbock ◽  
Anuradha Ramoji ◽  
Marie-Thérèse Hopp ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protective Factor ◽  
Biomedical Application ◽  
Homology Model ◽  
Binding Pocket ◽  
Heme Binding ◽  
High Affinity ◽  
Erythrocyte Lysis ◽  
Peptide Models ◽  
Displacement Assays

AbstractIn hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity in a 1:1 ratio. Here we show that hemopexin binds heme with lower affinity than previously assumed and that the interaction ratio tends to 2:1 (heme:hemopexin) or above. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. In addition, in silico molecular modelling with a newly created homology model of human hemopexin allowed us to propose a recruiting mechanism by which heme consecutively binds to several histidine residues and is finally funnelled into the high-affinity binding pocket. Our findings have direct implications for the biomedical application of hemopexin and its potential administration in hemolytic disorders.

Revisiting the interaction of heme with hemopexin

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Milena Sophie Detzel ◽  
Benjamin Franz Schmalohr ◽  
Francèl Steinbock ◽  
Marie-Thérèse Hopp ◽  
Anuradha Ramoji ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protective Factor ◽  
Homology Model ◽  
Binding Pocket ◽  
Heme Binding ◽  
Vis Spectroscopy ◽  
Erythrocyte Lysis ◽  
Peptide Models ◽  
High Concentrations ◽  
Displacement Assays

Abstract In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a K D value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.

scholarly journals Anti-diabetic drug binding site in a mammalian KATP channel revealed by Cryo-EM

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Gregory M Martin ◽  
Balamurugan Kandasamy ◽  
Frank DiMaio ◽  
Craig Yoshioka ◽  
Show-Ling Shyng
Keyword(s):  
Binding Sites ◽  
Katp Channel ◽  
Katp Channels ◽  
Binding Pocket ◽  
Drug Binding ◽  
Channel Activity ◽  
Nucleotide Binding Domain ◽  
High Affinity ◽  
Drug Binding Site ◽  
First Time

Sulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of a hamster SUR1/rat Kir6.2 channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.63 Å resolution, which reveals unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

scholarly journals Anti-diabetic drug binding site in KATP channels revealed by Cryo-EM

2017 ◽  
Author(s):  
Gregory M. Martin ◽  
Balamurugan Kandasamy ◽  
Frank DiMaio ◽  
Craig Yoshioka ◽  
Show-Ling Shyng
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Katp Channels ◽  
Binding Pocket ◽  
Drug Binding ◽  
Channel Activity ◽  
Nucleotide Binding Domain ◽  
High Affinity ◽  
Drug Binding Site ◽  
First Time

AbstractSulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of the channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.8Å resolution, which reveals in unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

scholarly journals In Silico Identification of Cholesterol Binding Motifs in the Chemokine Receptor CCR3

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 570
Author(s):  
Evan van van Aalst ◽  
Jotham Koneri ◽  
Benjamin J. Wylie
Keyword(s):  
Chemokine Receptor ◽  
Binding Sites ◽  
In Silico ◽  
Chemokine Receptors ◽  
Homology Model ◽  
Binding Pocket ◽  
Coarse Grained ◽  
Class A ◽  
Inflammatory Conditions ◽  
Cholesterol Binding

CC motif chemokine receptor 3 (CCR3) is a Class A G protein-coupled receptor (GPCR) mainly responsible for the cellular trafficking of eosinophils. As such, it plays key roles in inflammatory conditions, such as asthma and arthritis, and the metastasis of many deadly forms of cancer. However, little is known about how CCR3 functionally interacts with its bilayer environment. Here, we investigate cholesterol binding sites in silico through Coarse-Grained Molecular Dynamics (MD) and Pylipid analysis using an extensively validated homology model based on the crystal structure of CCR5. These simulations identified several cholesterol binding sites containing Cholesterol Recognition/Interaction Amino Acid Consensus motif (CRAC) and its inversion CARC motifs in CCR3. One such site, a CARC site in TM1, in conjunction with aliphatic residues in TM7, emerged as a candidate for future investigation based on the cholesterol residency time within the binding pocket. This site forms the core of a cholesterol binding site previously observed in computational studies of CCR2 and CCR5. Most importantly, these cholesterol binding sites are conserved in other chemokine receptors and may provide clues to cholesterol regulation mechanisms in this subfamily of Class A GPCRs.

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.

scholarly journals Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 164
Author(s):  
Lina Son ◽  
Elena Kryukova ◽  
Rustam Ziganshin ◽  
Tatyana Andreeva ◽  
Denis Kudryavtsev ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Gabaa Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine ◽  
High Affinity ◽  
Central Loop ◽  
Acetylcholine Binding Proteins ◽  
Α7 Nachrs

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

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