Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Abstract

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action. [1][2][3] One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers. 4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association. 6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve, 8 direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays. 9 The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay. [10][11][12][13] The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where β-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for β-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2). Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=2721 Protocol Cloning ConsiderationsThe TMD sequence should be 12-24 residues (shorter sequences wi...

Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1

“…To demonstrate the specificity of 1 , we tested its inhibitory effect on the diacylglycerol kinase (DAGK), a well studied 3-pass integral membrane protein that, like LMP-1, also forms a homo-trimer complex through TMD association [11]. As shown in Figure 3c, compound 1 did not inhibit DAGK TMD oligomerization, confirming the specificity of 1 to TMD-5.…”

“…We utilized a previously established ToxR assay as the platform for screening TMD disruptors in cellular membranes (Figure 3a) [11,12,13]. This E. coli -based assay uses a chimeric construct made of the N-terminal DNA binding domain of ToxR and the monomeric anchor maltose binding protein (MBP) fused to the LMP-1 TMD-5 cytoplasmic and periplasmic termini, respectively.…”

“…ToxR7-TMD-5, ToxR7–TMD of diacylglycerol kinase (DAGK) and ToxR7-TMD of integrin αIIb plasmids were constructed as described previously [11,12,13]. Diversity Set II library (1364 compounds) was provided by the Developmental Therapeutic Program, NCI/NIH.…”

“…ToxR7-TMD-5 plasmid (200 ng) was transformed into 200 μL of FHK12 competent cells (kindly provided by D. Langosch, Technische Universität München, Germany) with heat shock at 42 °C for 90 s and incubation on ice for 2 minutes, followed by addition of 800 μL SOC media and incubation with shaking at 37 °C for 1 h. 5 μL of the transformation mixture was used to inoculate 100 μL LB + arabinose (0.0025%) and chloramphenicol (30 μg/mL) and 100 μM compound. Cultures were incubated with shaking at 37 °C for 20 h and β-galactosidase activity was measured using a Beckman Coulter DTX 880 plate reader (Beckman, Coulter, Fullerton, CA, USA) as described previously [11,12]. Briefly, 5 μL of culture was transferred to the wells of a Costar 3596 polystyrene 96-well plate (Corning, NY, USA) containing 100 μL Z buffer/chloroform (1% β-mercaptoethanol, 10% chloroform, 89% A buffer: 1 M sodium phosphate, 10 mM KCl, 1 mM MgSO4 and pH 7.0).…”

Multi-Tox: Application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization

“…LMP-1 is a viral transforming protein essential for B cell immortalization by Epstein-Barr Virus (EBV) [16], playing a key role in EBV-dependent malignancies [17, 18]. The six-pass transmembrane domain of LMP-1 plays a critical role in the aberrant signaling of this protein [19]; intermolecular hydrogen bonding of a buried aspartic acid residue (D150) in TM5 drives oligomerization to form a constitutively activated complex [20, 21]. Although self-association of LMP-1 via its TMD is documented [19], the native oligomeric state of the protein has yet to be determined.…”