Reconstitution Studies Using the Helical and Carboxy-Terminal Domains of Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System

Biochemistry ◽  
1999 ◽  
Vol 38 (47) ◽  
pp. 15470-15479 ◽  
Author(s):  
Peng-Peng Zhu ◽  
Roman H. Szczepanowski ◽  
Neil J. Nosworthy ◽  
Ann Ginsburg ◽  
Alan Peterkofsky
Keyword(s):  
Phosphotransferase System ◽  
Carboxy Terminal ◽  
Enzyme I ◽  
Terminal Domains

scholarly journals Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions

2003 ◽  
Vol 17 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Raj Kumar ◽  
E. Brad Thompson
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Hormone Receptors ◽  
Nuclear Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Protein Interactions ◽  
Carboxy Terminal ◽  
And Function ◽  
Terminal Domains

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

Molecular map of the desmosomal plaque

1999 ◽  
Vol 112 (23) ◽  
pp. 4325-4336 ◽  
Author(s):  
A.J. North ◽  
W.G. Bardsley ◽  
J. Hyam ◽  
E.A. Bornslaeger ◽  
H.C. Cordingley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intermediate Filaments ◽  
Protein Interactions ◽  
Immunogold Labelling ◽  
Molecular Approaches ◽  
Molecular Map ◽  
Carboxy Terminal ◽  
Desmoglein 3 ◽  
Terminal Domains

Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the ‘a’ form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter ‘b’ form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

scholarly journals A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

2021 ◽  
Vol 8 ◽  
Author(s):  
Jeffrey A. Purslow ◽  
Jolene N. Thimmesch ◽  
Valeria Sivo ◽  
Trang T. Nguyen ◽  
Balabhadra Khatiwada ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Single Point ◽  
Conformational Dynamics ◽  
Phosphorylation Site ◽  
Phosphoryl Group ◽  
Single Point Mutation ◽  
Phosphotransferase System ◽  
Function Relationship ◽  
Enzyme I

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

scholarly journals Alpha-helix in the carboxy-terminal domains of histones H1 and H5.

1988 ◽  
Vol 7 (1) ◽  
pp. 69-75 ◽  
Author(s):  
D. J. Clark ◽  
C. S. Hill ◽  
S. R. Martin ◽  
J. O. Thomas
Keyword(s):  
Alpha Helix ◽  
Carboxy Terminal ◽  
Terminal Domains

Polymerization of Antarctic Fish Tubulins at Low Temperatures: Role of Carboxy-Terminal Domains

Biochemistry ◽  
1994 ◽  
Vol 33 (51) ◽  
pp. 15389-15396 ◽  
Author(s):  
William D. Singer ◽  
Sandra K. Parker ◽  
Richard H. Himes ◽  
H. William Detrich
Keyword(s):  
Low Temperatures ◽  
Antarctic Fish ◽  
Carboxy Terminal ◽  
Terminal Domains

scholarly journals Structural insight into glucose repression of the mannitol operon

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mangyu Choe ◽  
Huitae Min ◽  
Young-Ha Park ◽  
Yeon-Ran Kim ◽  
Jae-Sung Woo ◽  
...  
Keyword(s):  
Glucose Repression ◽  
Phosphorylation Site ◽  
Phosphotransferase System ◽  
E Coli ◽  
Carbon Compounds ◽  
Structural Insight ◽  
Enzyme I ◽  
Insight Into ◽  
Repressor Activity

Abstract Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within  the Enterobacteriaceae family.

A novel phosphoprotein dependent on the bacterial phosphoenolpyruvate–sugar phosphotransferase system

1983 ◽  
Vol 61 (2-3) ◽  
pp. 150-153 ◽  
Author(s):  
E. Bruce Waygood ◽  
Roshan L. Mattoo
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Growth Conditions ◽  
Phosphotransferase System ◽  
Crude Extracts ◽  
Enzyme I ◽  
Novel Protein

A protein has been found by isoelectricfocusing and autoradiography in Escherichia coli and Salmonella typhimurium which was phosphorylated by enzyme I and an histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate–sugar phosphotransferase system (PTS). This protein was not factor IIIglc nor was it specifically induced by fructose. Its presence in soluble crude extracts was dependent upon growth conditions; however, the two bacteria had different patterns and amounts in respect to this novel protein. The protein was present in S. typhimurium SB2950 which has an extensive deletion through the pts operon, thus indicating that it must be coded for elsewhere on the genome.

scholarly journals Interaction with Enzyme IIBMpo(EIIBMpo) and Phosphorylation by Phosphorylated EIIBMpoExert Antagonistic Effects on the Transcriptional Activator ManR of Listeria monocytogenes

2015 ◽  
Vol 197 (9) ◽  
pp. 1559-1572 ◽  
Author(s):  
Arthur Constant Zébré ◽  
Francine Moussan Aké ◽  
Magali Ventroux ◽  
Rose Koffi-Nevry ◽  
Marie-Françoise Noirot-Gros ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Glucose Sensor ◽  
Dual Role ◽  
Transcription Activator ◽  
Phosphotransferase System ◽  
Content Type ◽  
Antagonistic Effects ◽  
Virulence Gene Expression ◽  
Low Efficiency ◽  
Terminal Domains

ABSTRACTListeriae take up glucose and mannose predominantly through a mannose class phosphoenolpyruvate:carbohydrate phosphotransferase system (PTSMan), whose three components are encoded by themanLMNgenes. The expression of these genes is controlled by ManR, a LevR-type transcription activator containing two PTS regulation domains (PRDs) and two PTS-like domains (enzyme IIAMan[EIIAMan]- and EIIBGat-like). We demonstrate here that inListeria monocytogenes, ManR is activated via the phosphorylation of His585 in the EIIAMan-like domain by the general PTS components enzyme I and HPr. We also show that ManR is regulated by the PTSMpoand that EIIBMpoplays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIBMpointeracts with the two C-terminal domains of ManR (EIIBGat-like and PRD2) and that this interaction is required for ManR activity. Second, in the absence of glucose/mannose, phosphorylated EIIBMpo(P∼EIIBMpo) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of P∼EIIBMpoand P∼PRD2 of ManR, which together lead to the induction of themanLMNoperon. Complementation of a ΔmanRmutant with variousmanRalleles confirmed the antagonistic effects of PTS-catalyzed phosphorylation at the two different histidine residues of ManR. Deletion ofmanRprevented not only the expression of themanLMNoperon but also glucose-mediated repression of virulence gene expression; however, repression by other carbohydrates was unaffected. Interestingly, the expression ofmanLMNinListeria innocuawas reported to require not only ManR but also the Crp-like transcription activator Lin0142. Unlike Lin0142, theL. monocytogeneshomologue, Lmo0095, is not required formanLMNexpression; its absence rather stimulatesmanexpression.IMPORTANCEListeria monocytogenesis a human pathogen causing the foodborne disease listeriosis. The expression of most virulence genes is controlled by the transcription activator PrfA. Its activity is strongly repressed by carbohydrates, including glucose, which is transported intoL. monocytogenesmainly via a mannose/glucose-specific phosphotransferase system (PTSMan). Expression of themanoperon is regulated by the transcription activator ManR, the activity of which is controlled by a second, low-efficiency PTS of the mannose family, which functions as glucose sensor. Here we demonstrate that the EIIBMpocomponent plays a dual role in ManR regulation: it inactivates ManR by phosphorylating its His871 residue and stimulates ManR by interacting with its two C-terminal domains.

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