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Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Published on: May 26, 2011

Death Receptor 5 Activation Is Energetically Coupled to Opening of the Transmembrane Domain Dimer

Nagamani Vunnam¹, Cecily Kristine Campbell-Bezat¹, Andrew K Lewis¹

¹Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota.

Biophysical Journal

|July 27, 2017

Abstract:

The precise mechanism by which binding of tumor necrosis factor ligands to the extracellular domain of their corresponding receptors transmits signals across the plasma membrane has remained elusive. Recent studies have proposed that activation of several tumor necrosis factor receptors, including Death Receptor 5, involves a scissorlike opening of the disulfide-linked transmembrane (TM) dimer. Using time-resolved fluorescence resonance energy transfer, we provide, to our knowledge, the first direct biophysical evidence that Death Receptor 5 TM-dimers open in response to ligand binding. Then, to probe the importance of the closed-to-open TM domain transition in the overall energetics of receptor activation, we designed point-mutants (alanine to phenylalanine) in the predicted, tightly packed TM domain dimer interface. We hypothesized that the bulky residues should destabilize the closed conformation and eliminate the ∼3 kcal/mol energy barrier to TM domain opening and the ∼2 kcal/mol energy difference between the closed and open states, thus oversensitizing the receptor. To test this, we used all-atom molecular dynamics simulations of the isolated TM domain in explicit lipid bilayers coupled to thermodynamic potential of mean force calculations. We showed that single point mutants at the interface altered the energy landscape as predicted, but were not enough to completely eliminate the barrier to opening. However, the computational model did predict that a double mutation at i, i+4 positions at the center of the TM domain dimer eliminates the barrier and stabilizes the open conformation relative to the closed. We tested these mutants in cells with time-resolved fluorescence resonance energy transfer and death assays, and show remarkable agreement with the calculations. The single mutants had a small effect on TM domain separation and cell death, whereas the double mutant significantly increased the TM domain separation and more than doubled the sensitivity of cells to ligand stimulation.

Insights

Tumor necrosis factor receptor activation involves transmembrane (TM) dimer opening. This study provides biophysical evidence for Death Receptor 5 TM-dimer opening upon ligand binding, crucial for cell signaling.

Area of Science:

Biophysics
Molecular Biology
Cell Signaling

Background:

The precise mechanism of signal transduction by tumor necrosis factor (TNF) receptors upon ligand binding remains unclear.
Activation of some TNF receptors, like Death Receptor 5 (DR5), is proposed to involve a scissor-like opening of their transmembrane (TM) dimer.
Understanding this TM domain transition is key to elucidating receptor activation pathways.

Purpose of the Study:

To provide direct biophysical evidence for the opening of DR5 TM-dimers upon ligand binding.
To investigate the energetic landscape of the TM domain transition using computational and experimental approaches.
To determine the role of TM domain dimer interface mutations in receptor activation and cellular response.

Main Methods:

Time-resolved fluorescence resonance energy transfer (TR-FRET) to monitor TM-dimer separation in real-time.
All-atom molecular dynamics (MD) simulations of the isolated TM domain in lipid bilayers.
Thermodynamic potential of mean force (PMF) calculations to determine energy barriers.
Site-directed mutagenesis (alanine to phenylalanine) of the TM domain dimer interface.
Cell-based death assays to assess receptor sensitivity.

Main Results:

Direct biophysical evidence confirmed that DR5 TM-dimers open in response to ligand binding.
Computational simulations predicted that specific mutations at the TM dimer interface alter the energy landscape of opening.
Single point mutations partially destabilized the closed conformation but did not eliminate the opening barrier.
A double mutation at the center of the TM dimer interface was predicted to eliminate the opening barrier and stabilize the open state.
Experimental validation showed that the double mutant significantly increased TM-dimer separation and enhanced cellular sensitivity to ligand stimulation.

Conclusions:

Ligand binding induces a conformational change involving the opening of the Death Receptor 5 transmembrane dimer.
The transmembrane domain dimer interface plays a critical role in the energetics of receptor activation.
Targeted mutations can modulate the TM domain opening process, impacting receptor sensitivity and downstream signaling.
This study provides a mechanistic link between TM domain dynamics and TNF receptor signaling efficacy.

Read full analysis

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