Comparative Structural, Biophysical, and Receptor Binding Study of True Type and Wild type AAV2

2021 ◽  
pp. 107795
Author(s):  
Antonette Bennett ◽  
Joshua Hull ◽  
Nelly Jolinon ◽  
Julie Tordo ◽  
Katie Moss ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Study ◽  
Wild Type ◽  
True Type

scholarly journals Structure-function analysis of human transforming growth factor-α by site-directed mutagenesis

1992 ◽  
Vol 283 (1) ◽  
pp. 91-98 ◽  
Author(s):  
J A Feild ◽  
R H Reid ◽  
D J Rieman ◽  
T P Kline ◽  
G Sathe ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Receptor Binding ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Function Analysis ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Transforming Growth Factor Α

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

scholarly journals Correlation of the commercial anti-SARS-CoV-2 receptor binding domain antibody test with the chemiluminescent reduction neutralizing test and possible detection of antibodies to emerging variants

2021 ◽  
Author(s):  
Yoshitomo Morinaga ◽  
Hideki Tani ◽  
Yasushi Terasaki ◽  
Satoshi Nomura ◽  
Hitoshi Kawasuji ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Disease Onset ◽  
Antibody Test ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Healthy Individuals ◽  
Wild Type ◽  
Serum Samples ◽  
Serological Tests ◽  
Convalescent Phase

Background Serological tests are beneficial for recognizing the immune response against SARS-CoV-2. To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT), using pseudotyped SARS-CoV-2, is critical. Whether commercial antibody tests are comparably accurate is unknown. Methods Serum samples collected before variants were locally found were obtained from confirmed COVID-19 patients (n = 74), confirmed non-COVID-19 individuals (n = 179), and unscreened individuals (suspected healthy individuals, n = 229). The convalescent phase was defined as the period after day 10 from disease onset. The CRNT against pseudotyped viruses displaying the wild-type spike protein and a commercially available anti-receptor binding domain (RBD) antibody test were assayed. The CRNT was also assayed, using South African (SA) and United Kingdom (UK)-derived variants. Results The CRNT (cut off value, 50% inhibition) and the anti-RBD antibody test (cut off value, 0.8 U/mL) concurred regarding symptomatic COVID-19 patients in the convalescent phase and clearly differentiated between patients and suspected healthy individuals (sensitivity; 95.8% and 100%, specificity; 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titer (r = 0.47, 95% CI 0.20-0.68). Compared with the wild-type, CRNT reduction was observed for the SA and UK-derived variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the UK and the SA variants, respectively. Conclusion The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. The strong positive results using the commercial antibody test can reflect neutralizing activity against emerging variants.

Changes in angiotensin type 1 receptor binding and angiotensin-induced pressor responses in the rostral ventrolateral medulla of angiotensinogen knockout mice

2010 ◽  
Vol 298 (2) ◽  
pp. R411-R418 ◽  
Author(s):  
Daian Chen ◽  
Lisa Hazelwood ◽  
Lesley L. Walker ◽  
Brian J. Oldfield ◽  
Michael J. McKinley ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Receptor Binding ◽  
Knockout Mice ◽  
Pressor Response ◽  
Ventrolateral Medulla ◽  
Ang Ii ◽  
Wild Type ◽  
Intravenous Injections ◽  
Angiotensin Type

ANG II, the main circulating effector hormone of the renin-angiotensin system, is produced by enzymatic cleavage of angiotensinogen. The present study aimed to examine whether targeted deletion of the angiotensinogen gene ( Agt) altered brain ANG II receptor density or responsiveness to ANG II. In vitro autoradiography was used to examine the distribution and density of angiotensin type 1 (AT1) and type 2 receptors. In most brain regions, the distribution and density of angiotensin receptors were similar in brains of Agt knockout mice ( Agt −/− ) and wild-type mice. In Agt −/− mice, a small increase in AT1 receptor binding was observed in the rostral ventrolateral medulla (RVLM), a region that plays a critical role in blood pressure regulation. To examine whether Agt −/− mice showed altered responses to ANG II, blood pressure responses to intravenous injection (0.01–0.1 μg/kg) or RVLM microinjection (50 pmol in 50 nl) of ANG II were recorded in anesthetized Agt −/− and wild-type mice. Intravenous injections of phenylephrine (4 μg/kg and 2 μg/kg) were also made in both groups. The magnitude of the pressor response to intravenous injections of ANG II or phenylephrine was not different between Agt −/− and wild-type mice. Microinjection of ANG II into the RVLM induced a pressor response, which was significantly smaller in Agt −/− compared with wild-type mice (+10 ± 1 vs. +23 ± 4 mmHg, respectively, P = 0.004). Microinjection of glutamate into the RVLM (100 pmol in 10 nl) produced a robust pressor response, which was not different between Agt −/− and wild-type mice. A diminished response to ANG II microinjection in the RVLM of Agt −/− mice, despite an increased density of AT1 receptors suggests that signal transduction pathways may be altered in RVLM neurons of Agt −/− mice, resulting in attenuated cellular excitation.

scholarly journals GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

1993 ◽  
Vol 122 (6) ◽  
pp. 1253-1265 ◽  
Author(s):  
GW Kemble ◽  
YI Henis ◽  
JM White
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Binding Pocket ◽  
Binding Activity ◽  
Great Distance ◽  
Fusion Peptides ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Receptor Binding Activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.

scholarly journals Site directed mutants of human interleukin-1α: A 1 H-NMR and receptor binding study

FEBS Letters ◽  
1988 ◽  
Vol 231 (1) ◽  
pp. 135-138 ◽  
Author(s):  
Angela M. Gronenborn ◽  
Paul T. Wingfield ◽  
H.Robson McDonald ◽  
Ursula Schmeissner ◽  
G.Marius Clore
Keyword(s):  
Receptor Binding ◽  
Binding Study ◽  
H Nmr ◽  
Interleukin 1Α

scholarly journals An Antiviral Compound That Blocks Structural Transitions of Poliovirus Prevents Receptor Binding at Low Temperatures

2000 ◽  
Vol 74 (8) ◽  
pp. 3929-3931 ◽  
Author(s):  
Alan W. Dove ◽  
Vincent R. Racaniello
Keyword(s):  
Receptor Binding ◽  
Low Temperatures ◽  
Structural Changes ◽  
Cellular Receptor ◽  
Structural Transitions ◽  
Wild Type ◽  
Antiviral Compound

ABSTRACT Drugs such as WIN51711 that inhibit picornavirus replication are thought to block poliovirus infectivity by binding to the capsid and preventing structural transitions required for uncoating. We examined the activity of WIN51711 at temperatures where capsid flexibility is thought to be decreased. Below 37°C, WIN51711 inhibits the binding of wild-type poliovirus to cells but does not affect the binding of a poliovirus mutant which is believed to undergo structural transitions more readily. These results suggest that the poliovirus capsid must undergo structural changes to bind to its cellular receptor.

In vivo dopamine-D2 and serotonin-5-HT2 receptor binding study of risperidone and haloperidol

1994 ◽  
Vol 47 (3) ◽  
pp. 553-557 ◽  
Author(s):  
T. sumiyoshi ◽  
H. Kido ◽  
H. Sakamoto ◽  
K. Urasaki ◽  
K. Suzuki ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Study ◽  
Dopamine D2

scholarly journals Conserved in 186 countries the RBD fraction of SARS CoV-2 S-protein with in-silicoT500S mutation strongly blocks ACE2 rejecting the viral spike; A Molecular-docking analysis.

2021 ◽  
Author(s):  
Amrita Banerjee ◽  
Mehak Kanwar ◽  
Dipannita Santra ◽  
Smarajit Maiti
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Receptor Binding ◽  
Structural Alignment ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Docking Analysis ◽  
Global Pandemic ◽  
Complete Rejection ◽  
Molecular Docking Analysis

SARS-CoV-2 developed global-pandemic with millions of infections/deaths. Blocker/inhibitor of ACE2 and viral-spikes Receptor-Binding-Domain RBD-blockers are helpful. Here, conserved RBD (CUTs) from 186-countries were compared with WUHAN-Hu-1 wild-type by CLUSTAL-X2 and Structural-alignment using Pymol. The RBD of ACE2-bound nCOV2 crystal-structure (2.68)6VW1 was analyzed by Haddock-PatchDock. Extensive structural study/trial to introduce point/double/triple mutations in the following locations (Y489S/Y453S/T500S/T500Y)/(Y489S,Y453S/Y489S,T500S/Y489S,T500Y/Y453S,T500S/Y453S,T500Y)/ (Y489S,Y453S,T500S/Y489S,Y453S,T500Y) of CUT4 (most-effective) were tested with Swiss-Model-Expacy. Blind-docking of mutated-CUTs to ACE2 (6VW1) by Haddock-Hawkdock was performed and optimally complete-rejection of nCOV2 to ACE2 was noticed. Further, competitive-docking/binding-analyses were done by PRODIGY. Present results suggest that compared to the wild-spike, CUT4 showed extra LYS31-PHE490/GLN42-GLN498 bonding and lack of TYR41-THR500 interaction (in wild H-bond:2.639) with ACE2 RBD. Mutated-CUT4 strongly binds with the ACE2-RBD, promoting TYR41-T500S (H-bond: 2.0 and 1.8)/T500Y (H-bond:2.6) interaction and complete inhibition of ACE2 RBD-nCOV2. Mutant combinations T500S,Y489S,T500S and Y489S,Y453S,T500Y mostly blocked ACE2. Conclusively, CUT4-mutant rejects whole glycosylated-nCoV2 pre-dock/post-dock/competitive-docking conditions.

scholarly journals Epistasis at the SARS-CoV-2 RBD Interface and the Propitiously Boring Implications for Vaccine Escape

2021 ◽  
Author(s):  
Nash D. Rochman ◽  
Guilhem Faure ◽  
Yuri I. Wolf ◽  
Peter L. Freddolino ◽  
Feng Zhang ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Protein Structure Modeling ◽  
Escape Mutants ◽  
Small Set ◽  
Global Concern ◽  
The Impact

AbstractAt the time of this writing, August 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlap with the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple mutations in the RBD have rose to dominance. Non-additive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape is crucial. Epistasis can substantially increase the risk of vaccine escape and cannot be completely characterized through the study of the wild type (WT) alone. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the WT, Gamma (417T, 484K, 501Y), and Delta variants (452R, 478K). Overall, epistasis at the RBD surface appears to be limited and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2, whereas in the Gamma variant, epistasis more substantially destabilizes NAb interaction. These results suggest that the repertoire of potential escape mutations for the Delta variant is not substantially different from that of the WT, whereas Gamma poses a moderately greater risk for enhanced vaccine escape. Thus, the modest ensemble of mutations relative to the WT shown to reduce vaccine efficacy might constitute the majority of all possible escape mutations.SignificancePotential emergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild type SARS-CoV-2 virus as well as the Gamma and Delta variants. The results indicate that emergence of escape mutants is somewhat less likely for the Delta variant than for the wild type and moderately more likely for the Gamma variant. We conclude that the small set of escape-enhancing mutations already identified for the wild type is likely to include the majority of all possible mutations with this effect, a welcome finding.

scholarly journals An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Yan Guo ◽  
Wenhui He ◽  
Huihui Mou ◽  
Lizhou Zhang ◽  
Jing Chang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Binding Domain ◽  
Full Length ◽  
Vaccine Antigen ◽  
Receptor Binding Domain ◽  
Protein Vaccine ◽  
Wild Type ◽  
S Protein ◽  
Glycosylation Sites

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines. IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

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