scholarly journals Competitive displacement of full-length HIV-1 Nef from the Hck SH3 domain by a high-affinity artificial peptide

2007 ◽  
Vol 388 (6) ◽  
pp. 611-615 ◽  
Author(s):  
Thomas Stangler ◽  
Tuyen Tran ◽  
Silke Hoffmann ◽  
Holger Schmidt ◽  
Esther Jonas ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Phage Display ◽  
Dissociation Constant ◽  
Sh3 Domain ◽  
Competitive Displacement ◽  
Ligand Interaction ◽  
High Affinity ◽  
Hematopoietic Cell Kinase ◽  
Proline Rich Peptide ◽  
Hiv 1

AbstractWe studied the interaction of the artificial 12-aa proline-rich peptide PD1 with the SH3 domain of the hematopoietic cell kinase Hck and the peptide's potency in competitively displacing HIV-1 Nef from the Hck SH3 domain. PD1 was obtained from a phage display screen and exhibits exceptional affinity for the Hck SH3 domain (Kd=0.23 μM). Competition experiments using NMR spectroscopy demonstrate that the peptide even displaces Nef from Hck SH3 and allow for estimation of the Nef-Hck SH3 dissociation constant (Kd=0.44 μM), the strongest SH3 ligand interaction known so far. Consequences of this study for novel antiviral concepts are discussed.

HIV-1 Nef Alleles Show Differential Activation of Hematopoietic Cell Kinase in a Yeast Model System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3107-3107
Author(s):  
Ronald P. Trible ◽  
Thomas E. Smithgall
Keyword(s):  
Growth Inhibition ◽  
Mammalian Cells ◽  
Sh2 Domain ◽  
Model System ◽  
Hematopoietic Cell ◽  
Yeast Growth ◽  
High Affinity ◽  
Hematopoietic Cell Kinase ◽  
Aids Progression ◽  
Hiv 1

Abstract Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases that is expressed strongly in macrophages, an important target cell type for human immunodeficiency virus (HIV). The HIV Nef protein, a critical AIDS progression factor, binds Hck with unusually high affinity and induces Hck activation. Nef-mediated Hck activation has been proposed to trigger signaling pathways important for the establishment of HIV infection and subsequent progression to AIDS. Previous studies have utilized sequence alignments and binding assays to predict the functionality of Hck-Nef interactions, but these studies do not directly investigate the ability of Nef alleles to activate Hck. To better address this issue, we have created a model system in Saccharomyces cerevisiae based on previous findings that ectopic expression of c-Src arrests yeast growth in a kinase-dependent fashion. To establish the utility of this system for Hck and Nef, we first created a Hck variant containing a C-terminal sequence modified to encode the high-affinity SH2-domain binding sequence Tyr-Glu-Glu-Ile (HckYEEI). This modified sequence allows for autophosphorylation of the new tail and the subsequent intramolecular binding to the Hck SH2 domain. The resulting HckYEEI molecule models the physiologically down-regulated (inactive) form of Hck in structure and function. Unlike wild-type Hck, HckYEEI showed little kinase activity and failed to induce growth suppression in yeast. Importantly, co-expression with the Nef-SF2 allele activated HckYEEI, resulting in growth arrest. This effect was dependent on the conserved Nef proline-rich motif essential for Hck binding through its SH3 domain. However, Nef-SF2 did not activate FynYEEI, consistent with our previous data that the Fyn tyrosine kinase is not activated by Nef in mammalian cells. We then tested several additional laboratory HIV-1 Nef alleles for their ability to activate HckYEEI. Consensus, Lai, NL4.3, SF2, and YU-2 Nef alleles activated HckYEEI to varying degrees, as reflected by yeast growth inhibition. In contrast, Nef-Eli failed to suppress growth in the presence of HckYEEI, consistent with its inability to bind the Hck SH3 domain in vitro. Growth inhibition paralleled tyrosine phosphorylation of yeast proteins on anti-phosphotyrosine immunoblots. The differential effects of these Nef alleles on Hck activation correlated closely with previous results in mammalian cells. Taken together, these studies validate the use of yeast to reconstitute Nef-Hck interactions in a system amenable to screens of clinical Nef isolates, which may have predictive value in terms of AIDS progression.

scholarly journals A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

1995 ◽  
Vol 14 (20) ◽  
pp. 5006-5015 ◽  
Author(s):  
C. H. Lee ◽  
B. Leung ◽  
M. A. Lemmon ◽  
J. Zheng ◽  
D. Cowburn ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sh3 Domain ◽  
Single Amino Acid ◽  
High Affinity ◽  
Hiv 1

The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

1978 ◽  
Vol 40 (02) ◽  
pp. 350-357
Author(s):  
Robert H Yue ◽  
Menard M Gertler
Keyword(s):  
Molecular Weight ◽  
Dissociation Constant ◽  
Calcium Ions ◽  
Activation Process ◽  
Factor X ◽  
The Real ◽  
High Affinity ◽  
Binding Data ◽  
Sequential Mechanism ◽  
Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

In vitro binding activity between HCK SH3 domain and HIV-1 Nef protein

2011 ◽  
Vol 31 (3) ◽  
pp. 262-265
Author(s):  
Xiao-lin QIN ◽  
Chao-qi LIU ◽  
Dong-ming REN ◽  
Yong-qin ZHOU
Keyword(s):  
Sh3 Domain ◽  
Binding Activity ◽  
Hiv 1

scholarly journals HIV-1 Nef Selectively Activates Src Family Kinases Hck, Lyn, and c-Src through Direct SH3 Domain Interaction

2006 ◽  
Vol 281 (37) ◽  
pp. 27029-27038 ◽  
Author(s):  
Ronald P. Trible ◽  
Lori Emert-Sedlak ◽  
Thomas E. Smithgall
Keyword(s):  
Sh3 Domain ◽  
Src Family Kinases ◽  
Domain Interaction ◽  
Hiv 1

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library

AIDS ◽  
2004 ◽  
Vol 18 (2) ◽  
pp. 329-331 ◽  
Author(s):  
Sangeeta Karle ◽  
Stephanie Planque ◽  
Yasuhiro Nishiyama ◽  
Hiroaki Taguchi ◽  
Yong-Xin Zhou ◽  
...  
Keyword(s):  
Phage Display ◽  
Antibody Fragment ◽  
Phage Display Library ◽  
Hiv 1

scholarly journals Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
David Wensel ◽  
Yongnian Sun ◽  
Zhufang Li ◽  
Sharon Zhang ◽  
Caryn Picarillo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
High Affinity ◽  
Glycosylation Sites ◽  
Spectrum Activity ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Broad Spectrum Activity

ABSTRACT A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

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