Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. While normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein Latent Membrane Protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full length LMP-1 variant harboring the D150A substitution is deficient in NFκB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.

Increasing numbers of target protein structures available for computational studies makes the structure-based screening paradigm more attractive for initial hit indentification. We have developed a novel in silico screening methodology incorporating Molecular Mechanics (MM)/ implicit solvent methods to evaluate binding free energies and applied this technology to the identification of inhibitors of the TLR4/MD-2 interaction.* Corresponding author. Tel.: +1 303 492 6786; fax: +1 303 492 5894, hang.yin@colorado.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In order to improve affinity prediction accuracy without reducing screening speed, we developed a sequence of MM methods with implicit solvent models to evaluate binding free energies. Many recent investigations have revealed that similar methods, such as the MM/ Poisson Boltzman-Surface Area (MM/PB-SA) approach proved to be highly capable of predicting the binding free energies. NIH Public Access[23] To get through the bottleneck posed by the computationally demanding MD-based methods, we used a combination of the two: fast molecular docking for the generation of binding poses and MD simulations to rank the ligand poses according to their binding affinities implemented in QUANTUM. [23][24][25][26] To confine the amount of necessary calculations to a reasonable level, we performed extensive clustering of the ligand libraries to ensure the least possible computational work, while keeping as much of the full chemical diversity of the available library as possible. Finally, we conducted profiling of hits against a library of ca. 500 representative human proteins as a selectivity filter. As a proof of concept, screening was conducted against both TLR4 and MD-2 to validate our strategy and to maximize the potential of discovering a useful small molecule inhibitor. Compounds 1 and 2 were identified as potential TLR4-and MD-2-specific antagonists, respectively, with predicted K d values less than 10 μM (Figure 1).Because biophysical evaluation of TLR4 inhibitors is challenging due to the limited availability of TLR4 (recombinant expression of TLR4, an immune receptor that senses LPS, is not achievable in high-yielding bacterial systems), compounds 1 and 2 were tested in mammalian cells using a previously established TLR4 signaling assay. A challenge when developing TLR4 inhibitors is engineering selectivity against other TLRs. We investigated the selectivity of 1 by measuring nitric oxide (NO) production in RAW264.7 cells. RAW cells express all TLRs and each specific TLR can be individually activated by treatment w...

The efficient synthesis of a range of stable SAM mimetics, and their ability to promote the binding of the E. coli methionine repressor (MetJ) to its operator DNA, is described. Active analogues functionalised with bioorthogonal tags were identified.

ToxR-based transcriptional reporter assays allow the strength of transmembrane helix interactions in biological membranes to be measured. Previously, these assays have only been used to study single-pass transmembrane systems. To facilitate investigation of polytopic transmembrane domain (TMD) oligomerization, we applied the ToxR methodology to the study of multi-pass TMD oligomerization to give ‘Multi-Tox’. Association propensities of the viral oncoprotein, latent membrane protein-1 (LMP-1), and the E. coli membrane-integral diacylglycerol kinase (DAGK) were studied by Multi-Tox, highlighting residues of particular mechanistic importance. Both homo- and hetero-oligomerizations were studied.

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...