scholarly journals Identification of New In Vivo TonB-FepA Rendezvous Sites

2021 ◽  
Author(s):  
Kathleen Postle ◽  
Kelvin Kho ◽  
Michael Gresock ◽  
Joydeep Ghosh ◽  
Ray Larsen
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protonmotive Force ◽  
Amphipathic Helix ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Tonb System ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA β-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

scholarly journals Characterization of Escherichia coli ExbD protein modification in vivo

2020 ◽  
Author(s):  
Aruna Kumar ◽  
Kathleen Postle
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Active Transport ◽  
Protein Modification ◽  
Cytoplasmic Membrane ◽  
Physical Contact ◽  
Protonmotive Force ◽  
Iron Starvation ◽  
Tonb System

ABSTRACTThe TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

scholarly journals An ExbD Disordered Domain Peptide Inhibits TonB System Activity

2020 ◽  
Author(s):  
Dale R. Kopp ◽  
Kathleen Postle
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protonmotive Force ◽  
Gram Negative Bacteria ◽  
System Function ◽  
Gram Negative ◽  
Conserved Motif ◽  
Outer Membranes ◽  
Tonb System

ABSTRACTThe TonB system energizes transport of essential nutrients, such as iron siderophores, across unenergized outer membranes of Gram-negative bacteria. The integral cytoplasmic membrane proteins of the TonB system--ExbB, ExbD, and TonB--transduce the protonmotive force of the cytoplasmic membrane to TonB-dependent outer membrane transporters for active transport. ExbD protein is anchored in the cytoplasmic membrane, with the majority of it occupying the periplasm. We previously identified a conserved motif within a periplasmic disordered domain that is essential for TonB system function. Here we demonstrated that export of a peptide derived from that motif into the periplasm prevented TonB system function and inhibited all known ExbD interactions in vivo. Formaldehyde crosslinking captured the ExbD peptide in multiple ExbD and TonB complexes. Furthermore, peptides with mutations in the conserved motif not only had significantly reduced ability to inhibit TonB system activity, but they also altered interactions with ExbD and TonB, indicating the specificity of the interaction. Conserved motif peptide interactions with ExbD and TonB mostly occurred between Stage II and Stage III of the TonB energy transduction cycle, a transition that is characterized by the use of protonmotive force. Taken together, the data suggest that the ExbD disordered domain motif has multiple interactions with TonB and ExbD during between Stage II and III of the TonB energization cycle. Because of the essentiality of the motif, it may be a potential template for design of novel antibiotics that target the TonB system.IMPORTANCEGram-negative bacteria are intrinsically antibiotic-resistant due to the diffusion barrier posed by their outer membranes. The TonB system allows them to circumvent this barrier for their own nutritional needs, including iron. The ability of bacteria to acquire iron is a virulence factor for many Gram-negative pathogens. However, no antibiotics currently target the TonB system. Because TonB and ExbD must interact productively in the periplasm for transport across the outer membrane, they constitute attractive targets for potential antibiotic development where chemical characteristics need not accommodate the need to cross the hydrophobic cytoplasmic membrane. Here we show that a small ExbD-derived peptide can interfere with the TonB-ExbD interaction to inhibit the TonB system in vivo.

scholarly journals Both Carboxy- and Amino-Terminal Domains of the Vaccinia Virus Interferon Resistance Gene, E3L, Are Required for Pathogenesis in a Mouse Model

2001 ◽  
Vol 75 (2) ◽  
pp. 850-856 ◽  
Author(s):  
Teresa A. Brandt ◽  
Bertram L. Jacobs
Keyword(s):  
Cell Culture ◽  
Weight Loss ◽  
Mouse Model ◽  
Vaccinia Virus ◽  
Host Range ◽  
Broad Host Range ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

ABSTRACT The vaccinia virus (VV) E3L gene is responsible for providing interferon (IFN) resistance and a broad host range to VV in cell culture. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain, which is required for the IFN resistance and broad host range of the virus, has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA-dependent protein kinase, PKR. The amino-terminal domain, while conserved among orthopoxviruses, is dispensable in cell culture. To study the role of E3L in whole-animal infections, WR strain VV recombinants either lacking E3L (VVΔE3L) or expressing an amino-terminal (VVE3LΔ83N) or carboxy-terminal (VVE3LΔ26C) truncation of E3L were constructed. Whereas wild-type VV had a 50% lethal dose of approximately 104 PFU after intranasal infection, and elicited severe weight loss and morbidity, VVΔE3L was apathogenic, leading to no death, weight loss, or morbidity. VVΔE3L was also apathogenic after intracranial injection. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the amino- and carboxy-terminal domains of E3L were required for full pathogenesis in intranasal infections. These results demonstrate that the entire E3L gene is required for pathogenesis in the mouse model.

scholarly journals Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins.

1990 ◽  
Vol 111 (3) ◽  
pp. 1069-1079 ◽  
Author(s):  
M V de Arruda ◽  
S Watson ◽  
C S Lin ◽  
J Leavitt ◽  
P Matsudaira
Keyword(s):  
Calcium Binding ◽  
Actin Binding ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Actin Binding Domain ◽  
Solid Tissues ◽  
Complete Protein ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

scholarly journals Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210.

1993 ◽  
Vol 178 (6) ◽  
pp. 2237-2242 ◽  
Author(s):  
R E Nickowitz ◽  
H J Worman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Primary Biliary Cirrhosis ◽  
Antigenic Determinants ◽  
Nuclear Pore ◽  
Biliary Cirrhosis ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Patients with primary biliary cirrhosis (PBC) frequently have autoantibodies against a 210-kD integral glycoprotein of the nuclear envelope pore membrane. This protein, termed gp210, has a 1,783-amino acid amino-terminal domain located in the perinuclear space, a 20-amino acid transmembrane segment, and a 58-amino acid cytoplasmic carboxy-terminal tail. We now demonstrate that autoantibodies from 25 patients with PBC that recognize gp210 react with the cytoplasmic carboxy-terminal tail while none react with unmodified linear epitopes in the amino-terminal domain. The epitope(s) recognized by autoantibodies from all 25 patients is contained within a stretch of 15 amino acids. The recognized amino acid sequence is homologous to the protein products of the Escherichia coli mutY gene and Salmonella typhimurium mutB gene with an exact identity of six consecutive amino acids, suggesting that anti-gp210 antibodies may arise by molecular mimicry of bacterial antigenic determinants.

scholarly journals The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

FEBS Letters ◽  
2007 ◽  
Vol 581 (17) ◽  
pp. 3197-3203 ◽  
Author(s):  
Stephanie Herring ◽  
Alexandre Ambrogelly ◽  
Sarath Gundllapalli ◽  
Patrick O'Donoghue ◽  
Carla R. Polycarpo ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

scholarly journals 7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity.

1995 ◽  
Vol 129 (6) ◽  
pp. 1641-1650 ◽  
Author(s):  
X Zhu ◽  
I Lindberg
Keyword(s):  
Enzymatic Activity ◽  
Secretory Protein ◽  
Neuroendocrine Cells ◽  
Parent Cell ◽  
Intermediate Lobe ◽  
Amino Terminal ◽  
Highly Correlated ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

The prohormone convertase PC2, which is thought to mediate the proteolytic conversion of many peptide hormones, has recently been shown to interact with the neuroendocrine-specific polypeptide 7B2 in Xenopus intermediate lobe (Braks, J. A. M., and G. J. M. Martens. Cell. 78:263. 1994). In the present work we have stably transfected neuroendocrine cell lines with rat 7B2 constructs and found that overexpression of 27 kD 7B2 greatly facilitates the kinetics of maturation of proPC2, both in AtT-20/PC2 cells and in Rin5f cells. The half-life of conversion of proPC2 was reduced from 2.7 to 1.7 h in AtT-20/PC2 cells stably transfected with 27 kD 7B2 cDNA. The previously proposed "chaperone" domain was not sufficient for this facilitation event; however, a construct corresponding to the 21-kD 7B2 protein (which represents the naturally occurring maturation product) functioned well. A 7B2 construct in which maturation of 27 kD 7B2 to its 21-kD form was blocked was unable to facilitate maturation of proPC2. To correlate effects on PC2 maturation with the actual generation of PC2 enzymatic activity, a similar transfection of 21 kD 7B2 was performed using CHO cells previously amplified for the expression of proPC2. Enzymatic activity cleaving the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC was highly correlated with the expression of immunoreactive 21 kD 7B2 in the conditioned medium; medium obtained from the parent cell line was completely inactive. Enzymatic activity was identified as PC2 on the basis of inhibition by the carboxy-terminal peptide of 7B2, which has previously been shown to represent a potent and specific PC2 inhibitor. Taken together, our in vivo results indicate that the interesting secretory protein 7B2 is a bifunctional molecule with an amino-terminal domain involved in proPC2 transport as well as activation.

Sign in / Sign up

Export Citation Format

Share Document