A Vesicle Capture Sensor Chip for Kinetic Analysis of Interactions with Membrane-Bound Receptors

Cooper, Matthew A.; Hansson, Annelie; Löfås, Stefan; Williams, Dudley H.

doi:10.1006/abio.1999.4389

Cited by 176 publications

(180 citation statements)

References 32 publications

Supporting

Mentioning

175

Contrasting

Order By: Relevance

“…The ϳ32-kDa fragment was therefore identified as spanning Tir residues 1-314 (Fig. 4B) 28 Da different from that determined experimentally. The ϳ23-kDa fragment was therefore identified as spanning Tir residues 354 -550 (Fig.…”

contrasting

confidence: 57%

“…SPR can be used to investigate the interaction of proteins with lipid vesicles immobilized on a hydrophobic surface (26,27). The system employed utilized SUVs (composed of the inner leaflet mixture and pure PC) immobilized on a Pioneer L1 gold chip (28) as the ligand and FL-Tir (or individual domains) over a range of concentrations as the analyte. Changes in response as a result of protein binding were measured (Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Race

Lakey

Banfield

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Insertion of the enteropathogenic Escherichia coli Tir protein into the plasma membrane of intestinal epithelial cells is a crucial event in infection because it provides a receptor for intimate bacterial adherence. This interaction with the bacterial outer membrane protein intimin is also essential in generating a number of signaling activities associated with virulence. Tir can be modified at various sites by phosphorylation and functionally interacts with multiple host proteins. To investigate the mechanism of membrane insertion and to establish a model system in which the multiple interactions/ functions of Tir can be uncoupled and independently characterized, we used intrinsic tryptophan fluorescence, surface plasmon resonance, and protease digestion assays to show that Tir can insert directly into phospholipid vesicles in a composition-dependent manner to generate the topology reported in vivo. This is the first time that Tir has been shown to insert into membranes in a simple model system in the absence of chemical modification or other factors. These data are consistent with the protein interacting with lipids through two sites. The major site is localized to the transmembrane/intimin-binding domain region and includes Trp 235 , which is shown to be an effective reporter of interaction. The minor site is located within the C-terminal domain. Together, these data support a model in which Tir is released into the cytoplasm by the type III translocon and then independently inserts into the plasma membrane from a cytoplasmic location. A thorough understanding of this mechanism will be crucial to understand the subtleties of enteropathogenic E. coli pathogenesis. Enteropathogenic Escherichia coli (EPEC)3 is a major cause of infantile diarrheal disease in developing countries (1, 2) and is closely related to enterohemorrhagic E. coli (O157), which is an emerging health concern throughout the world (3). EPEC pathogenesis is dependent on (a) an active virulence-associated type III secretion system (TTSS) that directly injects "effector" proteins into host cells and (b) the interaction of Tir (translocated intimin receptor; one such effector protein), inserted into the host cell membrane, with the bacterial outer membrane protein intimin (recently reviewed in Refs. 4 and 5). The intimate adhesion of bacteria to host cell membranes as mediated by the intiminTir interaction is essential for virulence (6 -8) and results in subversion of host cell signaling processes promoting colonization of the intestinal tract (reviewed in Ref. 9).Following delivery by the TTSS, Tir inserts into the host cell plasma membrane, adopting a distinct topology composed of a large extracellular loop (the intimin-binding domain (IBD), residues 255-364) and N-terminal (residues 1-233) and C-terminal (residues 385-550) domains, which remain on the cytosolic face of the membrane (7, 10). These domains are linked by two predicted transmembrane helices (residues 234 -254 and 365-384). In this topology, the extracellular domain is available to ...

show abstract

contrasting

confidence: 57%

mentioning

confidence: 99%

Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Race

Lakey

Banfield

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Liposomes made of PC (neutral bilayer), PC:PBPS (1:1), PC:DOPG (1:1), and PC:PBPS:SDPS:DPPC (negatively charged bilayers) were injected and immobilized to the four flow cells on an L1 sensor chip, an ideal substrate for liposome adsorption (50,51). Liposomes were deposed on the chip at a low flow (Ͻ10 l/min) which has been found to provide a stable lipid surface (52,53).…”

Section: Binding Of Th-derived N-terminal Peptides and Of 14-3-3 Isofmentioning

confidence: 99%

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Halskau

Ying

Baumann

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylationdependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1-43)) and Ser 19 -phosphorylated (THp-(1-43)) states by surface plasmon resonance. THp-(1-43) showed high affinity for 14-3-3 proteins (K d ϳ 0.5 M for 14-3-3␥ and -and 7 M for 14-3-3 ). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S 0.5 ‫؍‬ 25-58 M (TH-(1-43)) and S 0.5 ‫؍‬ 135-475 M (THp-(1-43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3␥ showed a preferential binding to membranes, compared with 14-3-3 , both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3␥ for negatively charged membranes (S 0.5 ‫؍‬ 1-9 M) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser 19 -phosphorylated TH, 14-3-3␥, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of L-DOPA and dopamine synthesis.

show abstract

“…The increase in the magnitude of the signal is proportional to the concentration of the protein close to the surface of the sensor chip. We chose the L1 chip, which contains a dextran matrix with lipophilic alkyl chains that act as anchors to capture intact liposomes (37,43). In principle, this chip can also be used to study on-chip permeabilization of immobilized liposomes by membranedamaging agents (37).…”

Section: Binding Of Llo To Immobilized Liposomesmentioning

confidence: 99%

Sterol and pH Interdependence in the Binding, Oligomerization, and Pore Formation of Listeriolysin O

et al. 2007

View full text Add to dashboard Cite

Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3 -hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.

show abstract

A Vesicle Capture Sensor Chip for Kinetic Analysis of Interactions with Membrane-Bound Receptors

Cited by 176 publications

References 32 publications

Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Sterol and pH Interdependence in the Binding, Oligomerization, and Pore Formation of Listeriolysin O

Contact Info

Product

Resources

About