scholarly journals Specific cardiolipin–SecY interactions are required for proton-motive force stimulation of protein secretion

2018 ◽  
Vol 115 (31) ◽  
pp. 7967-7972 ◽  
Author(s):  
Robin A. Corey ◽  
Euan Pyle ◽  
William J. Allen ◽  
Daniel W. Watkins ◽  
Marina Casiraghi ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Transport ◽  
Membrane Lipids ◽  
Proton Motive Force ◽  
Proton Exchange ◽  
Motive Force ◽  
Coarse Grained ◽  
In Vitro Mutagenesis ◽  
Stimulation Of

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA–SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein–lipid interactions.

scholarly journals Specific cardiolipin-SecY interactions are required for proton-motive-force stimulation of protein secretion

2017 ◽  
Author(s):  
Robin A. Corey ◽  
Euan Pyle ◽  
William J. Allen ◽  
Marina Casiraghi ◽  
Bruno Miroux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Secretion ◽  
Binding Sites ◽  
Protein Transport ◽  
Proton Motive Force ◽  
Lipid Binding ◽  
Proton Exchange ◽  
Motive Force ◽  
Coarse Grained ◽  
Stimulation Of

AbstractThe transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin binding sites on the Thermotoga maritima SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry and biochemical analysis of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for cardiolipin in the conferral of PMF-stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery and thereby stimulate protein transport, by an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions.Significance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF- driven protein secretion.

scholarly journals In Vivo Synthesis of the Periplasmic Domain of TonB Inhibits Transport through the FecA and FhuA Iron Siderophore Transporters of Escherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 5885-5895 ◽  
Author(s):  
S. Peter Howard ◽  
Christina Herrmann ◽  
Chad W. Stratilo ◽  
V. Braun
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Outer Membrane Proteins ◽  
Proton Motive Force ◽  
Motive Force ◽  
In Vivo Studies ◽  
Siderophore Transport

ABSTRACT The siderophore transport activities of the two outer membrane proteins FhuA and FecA of Escherichia coli require the proton motive force of the cytoplasmic membrane. The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins. In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA. Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the fecABCDE transport genes by FecA, infection by phage φ80, and killing of cells by colicin M via FhuA. Transport of ferrichrome by FhuAΔ5-160 was also inhibited by TonB(33-239), although FhuAΔ5-160 lacks the TonB box which is involved in TonB binding. The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional. In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited. Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions.

In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly

2006 ◽  
Vol 275 (6) ◽  
pp. 540-552 ◽  
Author(s):  
Christiane Reinbothe ◽  
Frank Buhr ◽  
Sandra Bartsch ◽  
Claire Desvignes ◽  
Françoise Quigley ◽  
...  
Keyword(s):  
Binding Sites ◽  
Enzyme Catalysis ◽  
In Vitro Mutagenesis ◽  
Nadph:Protochlorophyllide Oxidoreductase

Stimulation of Plasmin Activity by Aspirin

1991 ◽  
Vol 65 (04) ◽  
pp. 389-393 ◽  
Author(s):  
Ariel Milwidsky ◽  
Zvendana Finci-Yeheskel ◽  
Michael Mayer
Keyword(s):  
Binding Sites ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Amidolytic Activity ◽  
Antithrombotic Effect ◽  
Plasmin Activity ◽  
Human Control ◽  
Significant Stimulation ◽  
Stimulation Of

SummaryThis study demonstrates an enhancing effect of aspirin on the amidolytic activity of plasmin. The stimulation of plasmin by aspirin was concentration-dependent and was attained at aspirin concentrations above 2 × 10−4 M. Aspirin produced a small, reproducible and statistically significant stimulation of the chromogenic activity of plasmin upon H-D-Valyl-L-Leucyl-L-Lysine-p-nitroanilide (3-2251) or pyro-Glu-Gly-Arg-p-nitroanilide (S-2444). Kinetic analysis demonstrated a slight decrease in the affinity of plasmin for substrate 3-2251 in the presence of aspirin, reflected by a change of the Km from 3.2 × 10-4 M to 3.8 × 10-4 M, and an increase of the Vm. The reciprocal Lineweaver-Burk curve indicated an uncompetitive type of stimulation. The stimulatory effect of aspirin was abolished by the lysine analogue α-aminohexanoic acid (AHA) but not by the α-amino acid glutamic acid. The effect of AHA suggests a specific involvement of lysine binding sites (LBS) on plasmin in the interaction of the enzyme with aspirin. Tlansient acidification of plasmin abolished its response to aspirin, to AHA and to their combination. The addition of aspirin to diluted human control or pregnancy plasma in vitro stimulated the plasma-mediated cleavage of the chromogenic substrate3-2251. In contrast to its effect on plasmin, aspirin failed to change the activity of tissue-type or urokinasetype plasminogen activators. It is conceivable that in addition to the antithrombotic effect of aspirin ascribed to its interaction with the platelets, aspirin also directly stimulates plasmin activity.

scholarly journals Dynamics of the localization of the plastid terminal oxidase inside the chloroplast

2020 ◽  
Vol 71 (9) ◽  
pp. 2661-2669 ◽  
Author(s):  
Susanne Bolte ◽  
Elodie Marcon ◽  
Mélanie Jaunario ◽  
Lucas Moyet ◽  
Maité Paternostre ◽  
...  
Keyword(s):  
Thylakoid Membrane ◽  
Fluorescent Protein ◽  
Proton Motive Force ◽  
Motive Force ◽  
Terminal Oxidase ◽  
Valve Function ◽  
Partial Dissociation ◽  
Green Fluorescent ◽  
Plastid Terminal Oxidase

Abstract The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOXs). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration, and setting the redox poise for cyclic electron transport. PTOX activity has been previously shown to depend on its localization at the thylakoid membrane. Here we investigate the dynamics of PTOX localization dependent on the proton motive force. Infiltrating illuminated leaves with uncouplers led to a partial dissociation of PTOX from the thylakoid membrane. In vitro reconstitution experiments showed that the attachment of purified recombinant maltose-binding protein (MBP)–OsPTOX to liposomes and isolated thylakoid membranes was strongest at slightly alkaline pH values in the presence of lower millimolar concentrations of KCl or MgCl2. In Arabidopsis thaliana overexpressing green fluorescent protein (GFP)–PTOX, confocal microscopy images showed that PTOX formed distinct spots in chloroplasts of dark-adapted or uncoupler-treated leaves, while the protein was more equally distributed in a network-like structure in the light. We propose a dynamic PTOX association with the thylakoid membrane depending on the presence of a proton motive force.

Multiple subelements within the polyomavirus enhancer function synergistically to activate DNA replication

1988 ◽  
Vol 8 (11) ◽  
pp. 5000-5015
Author(s):  
W J Muller ◽  
D Dufort ◽  
J A Hassell
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
T Antigen ◽  
Common Mechanism ◽  
In Vitro Mutagenesis ◽  
Large T Antigen ◽  
Enhancer Element ◽  
The Core ◽  
Beta Enhancer

The polyomavirus origin for DNA replication comprises at least two essential, but functionally distinct, cis-acting components. One of these, the origin core, is required only for DNA replication. It includes binding sites for large T antigen and the origin of bidirectional DNA replication. The other component is required for both transcription and DNA replication and is represented by two functionally redundant regions, alpha and beta, which are elements of the polyomavirus enhancer. Whereas either enhancer element will activate DNA replication, both enhancer elements are required to constitute a functional enhancer of transcription. To identify the sequences that make up each enhancer element, we have subjected them separately to in vitro mutagenesis and measured their capacity to activate replication in cis of the origin core in MOP-8 cells, which provide all trans-acting replicative functions including large T antigen. The results reveal that the beta enhancer element is composed of three subelements, two auxiliary subelements, and a core subelement. The core subelement independently activated DNA replication, albeit poorly. The auxiliary subelements, which were inactive on their own, acted synergistically with the core subelement to increase its activity. Interestingly, dimers of the beta core subelement functioned as well as the combination of a beta auxiliary subelement and a core subelement, suggesting that the subelements are functionally equivalent. The alpha enhancer element is organized similarly; it too comprises an auxiliary subelement and a core subelement. These results lead us to suggest that the polyomavirus enhancer comprises two levels of organization; two or more enhancer elements form an enhancer, and two or more subelements make up an enhancer element. The subelements share few sequences and serve as binding sites for distinct cellular factors. It appears, therefore, that a number of different cellular proteins function cooperatively to activate polyomavirus DNA replication by a common mechanism.

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