Structural Basis for Broad Detection of Genogroup II Noroviruses by a Monoclonal Antibody That Binds to a Site Occluded in the Viral Particle

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

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Preparation Of Factor IX-Deficient Human Plasma Using RFF-IX/1 Monoclonal Antibody

10.1055/s-0038-1653007 ◽

1981 ◽

Author(s):

F Rotblat ◽

A H Goodall ◽

G Janossy ◽

G Kemble ◽

D P O’Brien ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Line ◽

Factor Ix ◽

Hybrid Cells ◽

Spleen Cells ◽

High Affinity ◽

A Cell ◽

A Site ◽

Monoclonal Cell Line ◽

Mouse Myeloma

A cell line that secretes a monoclonal antibody to factor IX has been produced by fusing spleen cells from a mouse that had been hyper immunised to purified factor IX with mouse myeloma cells (line P3-NSI/I-Ag4-1). Hybrid cells were selected and a monoclonal cell line has been established in culture. This cell line secretes an IgGl(k) antibody (RFF-IX/1) with high affinity for a site related to the coagulant function of factor IX.Monoclonal antibody was partially purified from ascitic fluid from mice implanted with the RFF-IX/1 secreting cells by precipitation at 50% saturation with ammonium sulphate. This fraction has typically 630 NIH units/ml anti IX activity and 13.5 mg/ml protein. It was coupled to cyanogen bromide activated Sepharose 2B in the ratio of 9 mg. protein/1 ml gel. A column containing 10 ml of this gel removed all the assayable factor IX from the first 280 ml of normal ci.trated plasma that was passed over it. After that volume small amounts of factor IX could be detected in the effluent. Subsequently 10-20% of the factor IX activity adsorbed could be recovered by eluting the column with 3 M potassium iodide.Immuno-affinity depleted plasma could be used as substrate in a one-stage factor IX assay under routine laboratory conditions and was undistinguishable for that purpose from severe Christmas disease plasma.

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Structure-function relationships of stem cell factor: an analysis based on a series of human-murine stem cell factor chimera and the mapping of a neutralizing monoclonal antibody

Blood ◽

10.1182/blood.v88.2.437.bloodjournal882437 ◽

1996 ◽

Vol 88 (2) ◽

pp. 437-444 ◽

Cited By ~ 19

Author(s):

JV Matous ◽

K Langley ◽

K Kaushansky

Keyword(s):

Monoclonal Antibody ◽

Stem Cell ◽

Stem Cell Factor ◽

Functional Organization ◽

Biological Properties ◽

Structural Basis ◽

Gm Csf ◽

Neutralizing Monoclonal Antibody ◽

Macrophage Colony ◽

And Function

Although much is now known about the biological properties of the c-kit receptor and its ligand, stem cell factor (SCF), little is known of the structural basis for the binding and function of this hematopoietic cytokine. By analyzing the activities of chimeric interspecies and homologue muteins and epitope mapping of a monoclonal antibody (MoAb) to the human protein, we have found that three distinct regions of SCF are essential for full biological function. Homologue and interspecies swapping of polypeptide sequences between the amino terminus and G35, between L79 and N97, and between R121 and D128 reduced or eliminated the ability of the chimera to act in synergy with murine granulocyte- macrophage colony-stimulating factor (GM-CSF) to promote hematopoietic colony formation. Moreover, a nonconformation-dependent MoAb that neutralizes human, but not murine SCF, was found to bind to residues within the L79-N97 segment of the human homologue. As these three regions localize to the putative first, third, and fourth helices of the protein, findings remarkably similar to previous studies of cytokines as diverse as growth hormone, GM-CSF, and interleukin (IL)-4, our results suggest that cytokines of multiple classes share a common functional organization.

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Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Blood ◽

10.1182/blood.v99.4.1230 ◽

2002 ◽

Vol 99 (4) ◽

pp. 1230-1236 ◽

Cited By ~ 87

Author(s):

Zhong Q. Li ◽

Weiyi Liu ◽

Kwang S. Park ◽

Brue S. Sachais ◽

Gowthani M. Arepally ◽

...

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Binding Sites ◽

Heparin Therapy ◽

Common Complication ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

The Third ◽

N Terminus ◽

A Site

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.

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Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent

Structure ◽

10.1016/j.str.2017.12.010 ◽

2018 ◽

Vol 26 (2) ◽

pp. 187-198.e4 ◽

Cited By ~ 3

Author(s):

Lauren K. Ely ◽

Marco Lolicato ◽

Tovo David ◽

Kate Lowe ◽

Yun Cheol Kim ◽

...

Keyword(s):

Monoclonal Antibody ◽

Structural Basis ◽

Reversal Agent

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Structural basis of clade‐specific HIV‐1 neutralization by humanized anti‐V3 monoclonal antibody KD‐247

The FASEB Journal ◽

10.1096/fj.14-252262 ◽

2014 ◽

Vol 29 (1) ◽

pp. 70-80 ◽

Cited By ~ 1

Author(s):

Karen A. Kirby ◽

Yee Tsuey Ong ◽

Atsuko Hachiya ◽

Thomas G. Laughlin ◽

Leslie A. Chiang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Structural Basis ◽

Hiv 1

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A Novel Platelet Small-Molecule Agonist Targeting Glycoprotein Ibalpha.

Blood ◽

10.1182/blood.v114.22.4010.4010 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4010-4010

Author(s):

Jianfeng Yang ◽

Zhi Chen ◽

Weiliang Zhu ◽

Changgeng Ruan

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Small Molecule ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Critical Role ◽

Structural Basis ◽

Terminal Fragment ◽

Von Willebrand

Abstract Abstract 4010 Poster Board III-946 Introduction Interaction of glycoprotein (GP) Ibα with Von Willebrand factor (VWF) plays a critical role in platelet adhesion and signal transduction for αIIbβ3 activation under condition of high shear stress. Methods Based on the crystal structure of platelet GPIbα (PDB:1P9A), virtual screening was employed to identify active compounds. Compounds in SPECS database were docked to VWF binding site on the surface of GPIbα. The screening was carried out with the DOCK4.0 program. The 150 highest-scoring compounds were obtained for further bioassay and those with inhibitory activity of VWF binding to GPIbα were investigated the effect on platelet activation and aggregation. Results We found one compound, designated it as YC148, blocked ristocetin-induced plasma VWF binding to recombinant N-terminal fragment GPIbα (H1-V289) by ELISA method. More interestingly, YC148 did not inhibit ristocetin-induced platelet aggregation, on the contrary, it induced platelet aggregation itself in the absence of exogenous modulators such as ristocetin and botrocetin. A VWF A1 blocking antibody could not block platelet aggregation induced by YC148 despite it completely inhibited ristocetin-induced platelet agglutination. And YC148 also stimulated washed platelet aggregation where VWF was absent in the resuspension buffer. These indicated that the aggregation stimulated by YC148 could not the result from VWF binding. Flow cytomety also showed that YC148 increased P-selectin expression on platelet membrane and promoted monoclonal antibody PAC-1 binding to platelet. The platelet aggregation stimulated by YC148 was inhibited by anti-GPIbα monoclonal antibody AN51 and 6D1. Conclusion A novel exogenous small-molecule agonist was found to activate platelet through binding to GPIbα. It provides us a new tool for investigating platelet GPIb outside-in signaling pathway in platelet adhesion and aggregation. Furthermore, the structure of YC148 may provide a structural basis for developing new hemostatic drugs based on the inhibition of VWF-GPIb interaction. The effect of YC148 on platelet from Bernard-Soulier syndrome or GPIbα N-terminal fragment deficient platelet after in vitro cleavage will be further investigated. Disclosures: No relevant conflicts of interest to declare.

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Structural basis for the species selectivity of a fibrin-specific monoclonal antibody

Biochemistry ◽

10.1021/bi00354a039 ◽

1986 ◽

Vol 25 (6) ◽

pp. 1451-1455 ◽

Cited By ~ 10

Author(s):

Gary R. Matsueda ◽

Michael N. Margolies

Keyword(s):

Monoclonal Antibody ◽

Structural Basis ◽

Specific Monoclonal Antibody ◽

Species Selectivity

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Structural basis of adenylyl cyclase 9 activation

10.1101/2021.08.11.455989 ◽

2021 ◽

Author(s):

Chao Qi ◽

Pia Lavriha ◽

Ved Mehta ◽

Basavraj Khanppnavar ◽

Inayathulla Mohammed ◽

...

Keyword(s):

Secondary Structure ◽

Binding Site ◽

Adenylyl Cyclase ◽

Conformational Changes ◽

Structural Basis ◽

Structural Studies ◽

Nucleotide Analogue ◽

C Terminus ◽

Membrane Bound ◽

A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

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