scholarly journals Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain.

1994 ◽  
Vol 269 (36) ◽  
pp. 22524-22532 ◽  
Author(s):  
J.A. Silverman ◽  
J.A. Mindell ◽  
A. Finkelstein ◽  
W.H. Shen ◽  
R.J. Collier
Keyword(s):  
Diphtheria Toxin ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Helical Hairpin

scholarly journals Emergence of a new SARS-CoV-2 variant from GR clade with a novel S glycoprotein mutation V1230L in West Bengal, India

2021 ◽  
Author(s):  
Rakesh Sarkar ◽  
Ritubrita Saha ◽  
Pratik Mallick ◽  
Ranjana Sharma ◽  
Amandeep Kaur ◽  
...  
Keyword(s):  
South African ◽  
West Bengal ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Viral Envelope ◽  
Protein Coding ◽  
S Protein ◽  
Genomic Epidemiology ◽  
Clade B ◽  
New Variant

India is currently facing the devastating second wave of COVID-19 pandemic resulting in approximately 4000 deaths per day. To control this pandemic continuous mutational surveillance and genomic epidemiology of circulating strains is very important. In this study, we performed mutational analysis of the protein coding genes of SARS-CoV-2 strains (n=2000) collected during January 2021 to March 2021. Our data revealed the emergence of a new variant in West Bengal, India, which is characterized by the presence of 11 co-existing mutations including D614G, P681H and V1230L in S-glycoprotein. This new variant was identified in 70 out of 412 sequences submitted from West Bengal. Interestingly, among these 70 sequences, 16 sequences also harbored E484K in the S glycoprotein. Phylogenetic analysis revealed strains of this new variant emerged from GR clade (B.1.1) and formed a new cluster. We propose to name this variant as GRL or lineage B.1.1/S:V1230L due to the presence of V1230L in S glycoprotein along with GR clade specific mutations. Co-occurrence of P681H, previously observed in UK variant, and E484K, previously observed in South African variant and California variant, demonstrates the convergent evolution of SARS-CoV-2 mutation. V1230L, present within the transmembrane domain of S2 subunit of S glycoprotein, has not yet been reported from any country. Substitution of valine with more hydrophobic amino acid leucine at position 1230 of the transmembrane domain, having role in S protein binding to the viral envelope, could strengthen the interaction of S protein with the viral envelope and also increase the deposition of S protein to the viral envelope, and thus positively regulate virus infection. P618H and E484K mutation have already been demonstrated in favor of increased infectivity and immune invasion respectively. Therefore, the new variant having G614G, P618H, P1230L and E484K is expected to have better infectivity, transmissibility and immune invasion characteristics, which may pose additional threat along with B.1.617 in the ongoing COVID-19 pandemic in India.

scholarly journals Atypical Fusion Peptide of Nelson Bay Virus Fusion-Associated Small Transmembrane Protein

2005 ◽  
Vol 79 (3) ◽  
pp. 1853-1860 ◽  
Author(s):  
LiTing T. Cheng ◽  
Richard K. Plemper ◽  
Richard W. Compans
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Syncytium Formation ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Cell Surface Expression ◽  
Type I ◽  
Fusion Activity ◽  
Hydrophobic Domain

ABSTRACT A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.

scholarly journals The function of the transmembrane and cytoplasmic domains of pneumococcal penicillin-binding proteins 2x and 2b extends beyond that of simple anchoring devices

Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1585-1598 ◽  
Author(s):  
Kari Helene Berg ◽  
Daniel Straume ◽  
Leiv Sigve Håvarstein
Keyword(s):  
Binding Proteins ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Cytoplasmic Domain ◽  
Loss Of Function ◽  
Penicillin Binding Proteins ◽  
Catalytic Domains ◽  
Complex Process ◽  
Cytoplasmic Tails ◽  
Cross Links

The biosynthesis of cell-wall peptidoglycan is a complex process that involves six different penicillin-binding proteins (PBPs) in Streptococcus pneumoniae. Two of these, PBP2x and PBP2b, are monofunctional transpeptidases that catalyse the formation of peptide cross-links between adjacent glycan strands. Both of them are bitopic membrane proteins with a small cytoplasmic and a large extracellular domain. PBP2x and PBP2b are essential for septal and peripheral peptidoglycan synthesis, respectively. Although several studies have investigated the properties of their extracellular catalytic domains, it is not known whether the role of their N-terminal non-catalytic domains extends beyond that of being simple anchoring devices. We therefore decided to use reciprocal domain swapping and mutational analysis to gain more information about the biological function of the membrane anchors and cytoplasmic tails of PBP2x and PBP2b. In the case of PBP2x both domains are essential, but neither the membrane anchor nor the cytoplasmic domain of PBP2x appear to serve as major localization signals. Instead, our results suggest that they are involved in interactions with other components of the divisome. Mutations of conserved amino acids in the cytoplasmic domain of PBP2x resulted in loss of function, underlining the importance of this region. The cytoplasmic domain of PBP2b could be swapped with the corresponding domain from PBP2x, whereas replacement of the PBP2b transmembrane domain with the corresponding PBP2x domain gave rise to slow-growing cells with grossly abnormal morphology. When both domains were exchanged simultaneously the cells were no longer viable.

scholarly journals The transmembrane domain of diphtheria toxin improves molecular conjugate gene transfer

1997 ◽  
Vol 321 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Krishna J. FISHER ◽  
James M. WILSON
Keyword(s):  
Gene Transfer ◽  
Diphtheria Toxin ◽  
Transmembrane Domain ◽  
Transfection Efficiency ◽  
Bacterial Protein ◽  
Protein Fusion ◽  
Membrane Perturbation ◽  
Membrane Bound ◽  
Rna Transcript

Vectors based on the formation of a soluble DNA–polycation complex are being developed for the treatment of human diseases. These complexes are rapidly taken up by receptor-mediated endocytosis, but are inefficiently delivered to the nucleus owing to entrapment in membrane-bound vesicles. In this study we introduced the transmembrane domain of diphtheria toxin into a DNA–polycation conjugate complex in an effort to increase gene transfer by membrane perturbation. The transmembrane domain of diphtheria toxin was expressed in Escherichia coli as a maltose-binding protein fusion and chemically coupled to high-molecular-mass poly-l-lysine. Incorporation of this conjugate into a traditional complex formed with a luciferase-containing plasmid with an asialo-orosomucoid–polycation conjugate significantly increased transfection efficiency in vitro in a manner proportional to the amount of diphtheria toxin incorporated. The delivery of luciferase RNA transcript was similarly increased when complexed with similar polycation conjugates. This study uses the structural biology of a bacterial protein to improve polycation-based gene delivery.

scholarly journals Two Novel Fungal Phenolic UDP Glycosyltransferases from Absidia coerulea and Rhizopus japonicus

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Kebo Xie ◽  
Xiaoxiang Dou ◽  
Ridao Chen ◽  
Dawei Chen ◽  
Cheng Fang ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Molecular Level ◽  
Sequence Information ◽  
Content Type ◽  
C Terminus ◽  
Starting Point ◽  
Absidia Coerulea ◽  
Fungal Kingdom

ABSTRACT In the present study, two novel phenolic UDP glycosyltransferases (P-UGTs), UGT58A1 and UGT59A1, which can transfer sugar moieties from active donors to phenolic acceptors to generate corresponding glycosides, were identified in the fungal kingdom. UGT58A1 (from Absidia coerulea) and UGT59A1 (from Rhizopus japonicas) share a low degree of homology with known UGTs from animals, plants, bacteria, and viruses. These two P-UGTs are membrane-bound proteins with an N-terminal signal peptide and a transmembrane domain at the C terminus. Recombinant UGT58A1 and UGT59A1 are able to regioselectively and stereoselectively glycosylate a variety of phenolic aglycones to generate the corresponding glycosides. Phylogenetic analysis revealed the novelty of UGT58A1 and UGT59A1 in primary sequences in that they are distantly related to other UGTs and form a totally new evolutionary branch. Moreover, UGT58A1 and UGT59A1 represent the first members of the UGT58 and UGT59 families, respectively. Homology modeling and mutational analysis implied the sugar donor binding sites and key catalytic sites, which provided insights into the catalytic mechanism of UGT58A1. These results not only provide an efficient enzymatic tool for the synthesis of bioactive glycosides but also create a starting point for the identification of P-UGTs from fungi at the molecular level. IMPORTANCE Thus far, there have been many reports on the glycosylation of phenolics by fungal cells. However, no P-UGTs have ever been identified in fungi. Our study identified fungal P-UGTs at the molecular level and confirmed the existence of the UGT58 and UGT59 families. The novel sequence information on UGT58A1 and UGT59A1 shed light on the exciting and new P-UGTs hiding in the fungal kingdom, which would lead to the characterization of novel P-UGTs from fungi. Molecular identification of fungal P-UGTs not only is theoretically significant for a better understanding of the evolution of UGT families but also can be applied as a powerful tool in the glycodiversification of bioactive natural products for drug discovery.

scholarly journals Structural biology of the influenza virus fusion peptide.

2014 ◽  
Vol 61 (3) ◽  
Author(s):  
Remigiusz Worch
Keyword(s):  
Transmembrane Domain ◽  
Close Contact ◽  
Research Effort ◽  
Fusion Peptide ◽  
Viral Envelope ◽  
Fusion Peptides ◽  
Endosomal Membrane ◽  
Helical Hairpin ◽  
Large Conformational Change ◽  
Hemagglutinin Protein

The release of influenza RNA inside the host cell occurs through the fusion of two membranes, the viral envelope and that of the cellular endosome. The fusion is mediated by the influenza hemagglutinin protein (HA), in particular by the fusion peptide (HAfp) located in the N-terminal fragment of HA2 subunit. This protein fragment anchors in the internal endosomal membrane, whereas the C-terminal HA2 part comprises a transmembrane domain (TMD) embedded in the viral envelope. A drop of pH in the endosome acts as the main trigger for HA2 large conformational change that leads to anchoring of the fusion peptide, close contact of the membranes and the subsequent fusion. Throughout the years the major research effort was focused on a 20-amino acid fragment (HAfp1-20), shown by NMR to adopt a 'boomerang'-like structure. However, recent studies showed that extending HAfp1-20 by three highly conserved residues W21-Y22-G23 leads to formation of a unique, tight helical hairpin structure. This review summarizes recently discovered structural aspects of influenza fusion peptides and their relations with the membrane fusion mechanism.

scholarly journals Mutational Analysis of the Escherichia coli K-12 TolA N-Terminal Region and Characterization of Its TolQ-Interacting Domain by Genetic Suppression

1998 ◽  
Vol 180 (24) ◽  
pp. 6433-6439 ◽  
Author(s):  
Pierre Germon ◽  
Thierry Clavel ◽  
Anne Vianney ◽  
Raymond Portalier ◽  
Jean Claude Lazzaroni
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Membrane Integrity ◽  
Genetic Suppression ◽  
K 12

ABSTRACT The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.

Constitutive mutants of the GM-CSF receptor reveal multiple pathways leading to myeloid cell survival, proliferation, and granulocyte-macrophage differentiation

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 507-516 ◽  
Author(s):  
Anna L. Brown ◽  
Michelle Peters ◽  
Richard J. D'Andrea ◽  
Thomas J. Gonda
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Myeloid Cell ◽  
Proximal Region ◽  
Macrophage Differentiation ◽  
Gm Csf ◽  
Myeloid Cell Line ◽  
Constitutively Active Mutants ◽  
Macrophage Colony ◽  
Membrane Proximal Region

Abstract Activation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family of receptors promotes the survival, proliferation, and differentiation of cells of the myeloid compartment. Several signaling pathways are activated downstream of the receptor, however it is not clear how these induce specific biologic outcomes. We have previously identified 2 classes of constitutively active mutants of the shared signaling subunit, human (h) βc, of the human GM-CSF/interleukin-3 (IL-3)/IL-5 receptors that exhibit different modes of signaling. In a factor-dependent bipotential myeloid cell line, FDB1, an activated mutant containing a substitution in the transmembrane domain (V449E) induces factor-independent proliferation and survival, while mutants in the extracellular domain induce factor-independent granulocyte-macrophage differentiation. Here we have used further mutational analysis to demonstrate that there are nonredundant functions for several regions of the cytoplasmic domain with regard to mediating proliferation, viability, and differentiation, which have not been revealed by previous studies with the wild-type GM-CSF receptor. This unique lack of redundancy has revealed an association of a conserved membrane-proximal region with viability signaling and a critical but distinct role for tyrosine 577 in the activities of each class of mutant.

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