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

Nagamani Vunnam, Cecily Kristine Campbell-Bezat, Andrew K. Lewis, Jonathan N. Sachs

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)381-392
Number of pages12
JournalBiophysical journal
Issue number2
StatePublished - Jul 25 2017

Bibliographical note

Funding Information:
This work was supported by US National Institutes of Health R01 GM107175.

Publisher Copyright:
© 2017 Biophysical Society


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