Death Receptor 5 Activation Is Energetically Coupled to ......Article Death Receptor 5 Activation Is...

Article

Death Receptor 5 Activation Is EnergeticallyCoupled to Opening of the Transmembrane DomainDimer

Nagamani Vunnam,1 Cecily Kristine Campbell-Bezat,1 Andrew K. Lewis,1 and Jonathan N. Sachs1,*1Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota

ABSTRACT The precise mechanism by which binding of tumor necrosis factor ligands to the extracellular domain of their cor-responding receptors transmits signals across the plasma membrane has remained elusive. Recent studies have proposed thatactivation 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 theimportance 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 bulkyresidues should destabilize the closed conformation and eliminate the �3 kcal/mol energy barrier to TM domain opening andthe �2 kcal/mol energy difference between the closed and open states, thus oversensitizing the receptor. To test this, weused all-atom molecular dynamics simulations of the isolated TM domain in explicit lipid bilayers coupled to thermodynamic po-tential of mean force calculations. We showed that single point mutants at the interface altered the energy landscape as pre-dicted, but were not enough to completely eliminate the barrier to opening. However, the computational model did predictthat a double mutation at i, iþ4 positions at the center of the TM domain dimer eliminates the barrier and stabilizes the openconformation relative to the closed. We tested these mutants in cells with time-resolved fluorescence resonance energy transferand death assays, and show remarkable agreement with the calculations. The single mutants had a small effect on TM domainseparation and cell death, whereas the double mutant significantly increased the TM domain separation and more than doubledthe sensitivity of cells to ligand stimulation.

INTRODUCTION

Oligomerization of single-pass plasma membrane recep-tors is well established as a common structural mechanismfor activation (1–13). Significant questions remain as to if,and how, receptor oligomerization may be energeticallycoupled to conformational changes that transduce the signalacross the plasma membrane. A number of models havebeen proposed to explain this coupling, including ligand-dependent (14,15) and ligand-independent (16,17) mecha-nisms, although backbone conformational dynamics areoften absent in these models. In multiple superfamilies,receptor dimerization has been shown to involve weak,metastable interactions between transmembrane domain

Submitted March 29, 2017, and accepted for publication May 22, 2017.

*Correspondence: [email protected]

Nagamani Vunnam and Cecily Kristine Campbell-Bezat contributed

equally to this work.

Editor: Kalina Hristova.

http://dx.doi.org/10.1016/j.bpj.2017.05.038

� 2017 Biophysical Society.

(TM) a-helices (18–26). A recent and landmark study offibroblast growth factor receptors used Forster resonanceenergy transfer (FRET) to show that ligand binding inducessubtle conformational changes in the TM domain dimerthat correlate with changes in functional phosphorylation(27). In particular, ligand binding was shown to reduce thespacing between the TM domain monomers; existence ofmultiple interhelical separations suggested that receptorTM domains have evolved nuanced structural states thatsupport diverse function (27).

Although the free energy of TM domain dimerization hasbeen discussed at length (28–31), what has remained uncer-tain is the free energy difference between various conforma-tional states of TM domain dimers. In two members of theTNF Receptor (TNFR) superfamily, namely Death Receptor5 (DR5L) and tumor necrosis factor receptor 1 (TNFR1), werecently proposed that ligand binding changes the confor-mation of the extracellular domain (ECD) of preassembleddimers, which in turn propagates through the membrane

Biophysical Journal 113, 381–392, July 25, 2017 381

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Vunnam et al.

via opening of this TM dimer (Fig. 1 A) (6,32). We showedthat in DR5L, ligand binding causes the formation of araft-dependent, disulfide-linked TM domain dimer (33),and predicted a scissorslike opening with the disulfide link-age acting as the hinge. Our model was inspired by Vilaret al. (34) who proposed, but did not demonstrate, a similarmodel for activation of p75 neurotrophin receptor, whichalso has disulfide-linked TM-domain dimers.

Using computational modeling of DR5L, validated byexperiments on isolated TM a-helices, we first predicted aTM domain dimer structure, and then showed that thedimer can adopt two distinct structural states that are sepa-rated by an �3 kcal/mol energy barrier: 1) a tightly packed,low-energy closed state; and 2) a higher-energy, openstate, with a free energy difference between the two statesof DGTM

opening � 2 kcal/mol (35). We hypothesized that inthe full-length receptor these two TM dimeric states corre-spond to the inactive, preligand structure (closed), and tothe active, ligand bound structure (open). Low-resolutionacceptor-photobleaching FRET experiments on a disease-related, constitutively active mutant supported this model(32). However, there has been no direct experimental evi-

A

C

FIGURE 1 Activation ofDR5L involves a ligand-induced opening of the disulfi

DR5L activation. Cysteines are shown as yellow spheres. Spectral decay curves

pairs are given in the absence (B) and the presence (C) of TRAIL. Figures are re

efficiency in the presence and absence of TRAIL. Mean values and standard dev

382 Biophysical Journal 113, 381–392, July 25, 2017

dence in cells to corroborate the importance of ligand-induced conformational changes in the TM domain dimerof TNFRs. Likewise, there is no available experimental orcomputational data to suggest whether the weak forces hold-ing the TM domain dimer in the closed conformation aresubstantial enough to influence the overall sensitivity ofthe receptor to ligand.

Building on these DR5L and fibroblast growth factorreceptor studies, we wanted to know whether, based oncomputationally derived thermodynamic free energy pro-files, it is possible to rationally control receptor functionby altering the TM domain conformation in a predefinedmanner. Specifically, could we use mutagenesis at the pre-dicted dimer interface to either eliminate the 3 kcal/molfree energy barrier and/or decrease the relative free energyof the open state and thereby bias the TM domains ofDR5L to the open, active conformation? First, we neededto prove that ligand-induced activation is accompanied byan opening of the TM domain. We used live-cell, high-resolution lifetime measurements of FRET to provide thefirst direct biophysical evidence of the opening of theTM domain of DR5L upon ligand binding and receptor

B

D

de-linkeda-helical TMdomain dimer. (A) Given here is a proposedmodel for

of donor-only (DR5DDD/CFP) and donor-with-acceptor (DR5LDDD/YFP)

presentative of four independent experiments. (D) Given here is the FRET

iations of four experiments are plotted. To see this figure in color, go online.

Thermodynamics of Transmembrane Domains

activation. Second, we aimed to prove whether manipulatingthe free energy landscape of the TM domain’s dynamicclose-to-open transitions could sensitize the full-length re-ceptor to ligand stimulation. To determine the key aminoacids that dictate changes in TM-domain conformations,we investigated the effect of point mutations (A18F,A25F, and A18F/A25F) within the TM domain, validatingresults from potential-of-mean-force (PMF) calculationswith cell-based experiments. Following our previousapproach (35), the PMF calculations on the mutants weredone on isolated TM dimers within an all-atom, fully hy-drated lipid bilayer, to predict how these mutations mightperturb the open and closed energy landscape of DR5L.FRET-based and functional analysis of the mutants corrob-orate the computational predictions, showing that the pointmutations within the TM domain of DR5L can shift the pop-ulation distribution of TM-dimers toward the open confor-mation and significantly increase the apoptotic activity ofthe receptor.

MATERIALS AND METHODS

Cell cultures and reagents

HEK293 cells were cultured in phenol red-free DMEM (Gibco Labora-

tories, Gaithersburg, MD). BJAB-derived cells (36) were cultured in

RPMI 1640 with HEPES, sodium pyruvate, and L-glutamine (ATCC, Man-

assas, VA). All media were supplemented with 2 mML-Glutamine (Invitro-

gen, Carlsbad, CA), heat-inactivated 10% fetal bovine serum (FBS HI;

Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (HyClone,

Logan, UT). Cell cultures were maintained in an incubator with 5% CO2

(Forma Series II Water Jacket CO2 Incubator; Thermo Fisher Scientific,

Waltham, MA) at 37�C. Antibodies for Western blots, DR5 antibodies

(mAb6313), and fluorescent secondary antibody (NL637) were purchased

from R&D Systems (Minneapolis, MN). PE anti-human CD262 (DR5;

TRAIL-R2) antibody (DJR2-4 PE) was purchased from BioLegend (San

Diego, CA). b-Actin (ab8227) was purchased from Abcam (Cambridge,

UK). The N-terminal FLAG-tagged soluble TRAIL (residues 114–281)

was overexpressed using the pT7-FLAG-1 inducible expression system in

Escherichia coli and purified by anti-FLAG-affinity column (M2 anti-

FLAG-agarose resin; Sigma-Aldrich, St. Louis, MO).

Molecular biology

pECFP-N1 and pEYFP-N1 constructs were a kind gift from David D.

Thomas. EcoRI and BamHI restriction enzymes were used to clone

DR5DDD (1–240) into pECFP-N1 and pEYFP-N1 vectors. DR5DDD

(1–240) was inserted at the N-terminus of the ECFP and EYFP. To pre-

vent the clustering of fluorophores, we mutated alanine 206 to lysine

(A206K) in both ECFP and EYFP constructs. Complementary DNA for

full-length DR5L (residues 1–440) was cloned into pcDNA3.1(þ) vector

by using standard cloning protocol. All mutations were introduced by

QuikChange mutagenesis (Stratagene, San Diego, CA) and verified by

sequencing.

Live-cell time-resolved FRET measurements

HEK 293 cells were grown to 75% confluence in a 10 cm plate and tran-

siently transfected by the calcium phosphate method with either donor-

only (DR5LDDD/CFP) or a donor-acceptor pair (DR5LDDD/CFP and

DR5LDDD/YFP), at 1:5 donor/acceptor (total DNA 15 mg/plate). After

8–10 h, growth medium was replaced with fresh growth medium. Cells

were incubated for a further 24–48 h before being used for experiments.

Two days after transfection, cells were lifted with trypsin, washed three

times with PBS, and resuspended in �4 mL of PBS at a concentration of

1,000,000 cells/mL. For lifetime measurements, transfected cells were

dispensed (50 mL/well) into a 384-well glass-bottom plate by using multi-

drop combi-reagent dispenser (Thermo Fisher Scientific). Donor lifetime

in the presence and absence of acceptor was measured by using a fluores-

cence lifetime plate reader (Fluorescence Innovations, Minneapolis, MN).

CFP fluorescence was excited with a 473-nm microchip laser and emission

was filtered with 488-nm long-pass and 517/20-nm band-pass filters. Time-

resolved fluorescence waveforms for each well were fitted to single-expo-

nential decays using least-squares minimization global analysis software

(Fluorescence Innovations) to give donor lifetime (tD) and donor-acceptor

lifetime (tDA) measurements. FRET efficiency (E) was then calculated

based on the following:

E ¼ 1��tDAtD

�: (1)

PMF calculations

The starting configuration of the DR5L wild-type (WT) TM dimer was

previously generated through REMD (6). PMF calculations (37) were

performed on this dimer using the Leu-32/Leu-32 Ca distance as the re-

action coordinate. Residues were renumbered so that Thr-204 in full-

length DR5L corresponded to Thr-1 in the simulated helix. Cys-209

became Cys-6 and to generate starting configurations along the reaction

coordinate, the dimer was rotated through the disulfide dihedral angle.

Fifty initial configurations were generated this way in CHARMM-GUI

(38) with the angle increasing by 0.5� in each window. A 2 kcal/mol

per A2 harmonic restraint was applied using the NAMD Colvars module

(39) and each configuration was inserted into a DMPC bilayer and sol-

vated with TIP3 water. For the mutated constructs, point mutations

were made to the initial dimer construct in VMD and then simulations

and PMF calculations were carried out as in Humphrey et al. (37). The

extracellular domain of TNFR1 (PDB:1NCF) (40) was simulated using

residues 14–150 of chains A and B. The protein was solvated in

36,236 TIP3 (41) water molecules using CHARMM-GUI (38). Simula-

tions were carried out using NAMD 2.8 (39). The system was relaxed

for 10,000 steps using steepest descent minimization. Molecular dy-

namics were run using the NPT ensemble and the CHARMM36 force-

field parameters (42–44) at a temperature of 300 K and a pressure of

1 atm using the Langevin and Nos�e-Hoover methods (45), respectively.

Long-range electrostatics were calculated using the particle-mesh Ewald

method (46) with a 1.5 A grid spacing and fourth-order interpolation.

Lennard-Jones interactions were cut off at 10 A. The system was propa-

gated using the r-RESPA algorithm (47) with a 2-fs time step and cova-

lent bonds involving hydrogen were constrained using the RATTLE

algorithm (48). The system was equilibrated by running with Ca atoms

restrained at 1, 0.1, and 0.01 kcal/mol per A2 for 2 ns at each stage.

The PMF was generated using the adaptive biasing force (ABF) module

(49–51). We selected the distance between the centers of mass of the Ca

atoms in CRD4 (residues 120–150 in 1 ncf) in chains A and B as the re-

action coordinate to best represent opening of the membrane-proximal

extracellular domain. The lower and upper boundaries were set to 6

and 30 A, respectively, the bin width was set to 0.1 A, and the ABF

was calculated after collecting 10,000 steps in each bin. The upper and

lower boundaries were eventually adjusted to 17.5 and 6 A, respectively,

to restrict the conformational space to the interesting region of the PMF.

An additional window was run with the boundaries set to 11.5 and

15.5 A, then adjusted to 10.5 and 12.5 A, to improve sampling in that

region. We collected data from at least 5 � 105 steps for all bins between


Vunnam et al.

11 and 20 A. Trajectory analysis and snapshot rendering was performed

with the aid of the software programs LOOS (52) and VMD (37).

Western blotting

Cells were pelleted by centrifugation and lysed with RIPA buffer (Thermo

Fisher Scientific) supplemented with protease inhibitors for 30–60 min at

4�C. Total protein concentration of lysates was determined by BCA assay

(53) and equal amounts of total protein (�60 mg) were mixed with 4�Bio-Rad sample buffer (Bio-Rad, Hercules, CA) and boiled for 3–5 min.

Lysates were run on 4–15% Tris-glycine SDS-PAGE gels (Bio-Rad) and

blotted on a PVDF transfer membrane (EMD Millipore, Billerica, MA).

Primary antibodies against DR5 and b-Actin were used.

Cell viability and surface expressiondetermination

The effect of TM domain mutation on TRAIL-induced apoptosis was deter-

mined using an MTT assay. DR5-deficient BJAB cells (10,000,000/mL),

were transfected with WT and mutant plasmids by the Neon Transfection

System (Invitrogen), using a pulse voltage of 1350 and a pulse width of

20 ms. Electroporated cells (100 mL) were transferred into 10 mL growth

medium (10 cm plate) without antibiotics and incubated for 24 h at 37�C,5% CO2. Next day, live transfected cells were divided into two portions.

One portion of the cells was aliquoted onto a 96-well plate for a cell

viability assay and the other half was used to test the transfection efficiency.

To determine the effect of mutants on TRAIL-induced apoptosis, cells

were treated with TRAIL (0.1 mg/mL) and incubated further for 24 h

at 37�C. Cell viability was measured using an MTT assay. DR5 surface

expression was measured by antibody staining using PE anti-human

CD262 (DR5; TRAIL-R2) antibody. Surface expression levels were deter-

mined by measuring the fluorescence intensity using a fluorescence lifetime

plate reader (Fluorescence Innovations).

RESULTS

DR5L activation involves a ligand-inducedopening of the disulfide-linked TM domain dimer

Recent studies proposed that activation of DR5L involves ascissorlike opening of the TM-receptor dimer (Fig. 1 A).However, the opening of the DR5L TM-domain in responseto ligand binding has not been shown experimentally.To characterize the ligand-induced conformational changewithin the TM-domain, we removed the death domain(DD) of DR5L (DRL5DDD) and attached fluorescent tags(cyan or yellow fluorescent proteins, ECFP or EYFP,respectively) to the cytoplasmic end of the TM domain.Because the fluorescent proteins were attached directly tothe TM domains, we could directly monitor changes inthe dynamics of the TM domain in response to ligand bind-ing. It has been shown previously that the addition of afluorescent tag to this region of the protein does not affectthe assembly of DR5L (6). Time-resolved fluorescence reso-nance energy transfer (TR-FRET) experiments were carriedout in transiently transfected HEK293 cells coexpressingDR5LDDD-fused CFP and/or -YFP. Lifetime measurementsare largely independent of receptor concentration, whichmakes the transient transfection method a suitable one for


our experiment. Donor (DR5LDDD/CFP) lifetime in thepresence and absence of acceptor (DR5LDDD/YFP) wasmeasured and then used to calculate FRET efficiency.Measurements showed a substantial decrease in the fluores-cence lifetime of the donor in the presence of the acceptorcompared with the donor only (Fig. 1 B), which confirmsefficient energy transfer between the FRET pairs. Theseresults show that DR5LDDD exist as ligand-independentoligomers. We then evaluated the effect of TRAIL onDR5L TM domain conformations through FRET efficiency.Cells treated with TRAIL showed significantly lower FRET(16.4%) compared with untreated cells (25%) (Fig. 1, Cand D). Decrease in FRETefficiency shows that the distancebetween donor-acceptor pairs is increased in response toTRAIL binding, indicating a shift in the population distri-bution of the TM domain conformations toward an openconformation.

PMF calculations predict mutations at the TMdomain dimer interface should favor the openconformation

Next, we sought to determine the key motifs that control theconformational dynamics of DR5LTM-dimers. To this end,PMF calculations were performed on the isolated TM dimer.Point mutations were made in the predicted TM dimer inter-face (35) with the goal of perturbing the resulting energylandscape so that it sufficiently favored the open confor-mation. Previous PMF calculations had shown that theGxxxG motif was unperturbed in the opening of the WTsequence. Instead, the small, interfacial amino acids down-stream of the GxxxG were seen to be critical in establishingthe closed-to-open transition barrier. Thus, Alanine-18and -25 (A18 and A25) (Fig. 2 A) were chosen becausethey were tightly packed in the closed conformational tra-jectories of the WT PMF calculations. We mutated theseresidues to bulky phenylalanine residues with the expecta-tions of increasing unfavorable steric interactions betweenthe helices in the closed conformation. In addition to singlemutants, we also performed calculations on a double mutant(DM; A18/A25).

There are two relevant aspects of the free energy land-scape for TM opening: 1) the activation free energy, whichdetermines the kinetics of the transition between states;and 2) the difference in free energies between the openand closed conformations, which reflects the equilibriumdistribution of the two states. The free energy profile foreach of the mutant constructs is significantly altered ascompared to that predicted by the WT PMF calculations(35) (Fig. 2, B–E). The differences manifest in both the en-ergy barrier (which is lowered) and the relative free energyof the two states. The WT energy landscape (Fig. 2 B) has atightly defined closed conformation with a clear energy bar-rier (��3 kcal/mol; Table 1) separating it from a broaderensemble of open conformations. Additionally, in the WT,

FIGURE 2 PMF calculations predict mutations at the TM domain dimer interface favor the open conformation. (A) Shown here is the isolated transmem-

brane domain sequence of DR5L. Residues of interest are shown in red. Cysteine at sixth position participates in disulfide-linked dimerization. Rendering

illustrates ligand-induced opening of DR5LTM dimers as predicted from statistical mechanical calculations on the PMF trajectories of isolated TM domain

dimers of WT. Shown here are PMF calculations on WT (B), two single mutants (C and D), and a double mutant (E) isolated DR5 TM dimer along the

Leu-32/Leu-32 reaction coordinate. To see this figure in color, go online.


the closed-state energy is more favorable than the open-stateenergy, with a DGTM

opening � 2 kcal/mol. The single and dou-ble mutants successfully bias DR5L-DR5L interactions to-ward an open conformation by reducing both the energybarrier and the difference in energy between the two states.Statistical differences are given in Table 1. The minimalenergies of the two states are approximately equal forthe two single mutants (Fig. 2, C and D), implying thatDGTM

opening � 0 kcal/mol, and in each case the energybarrier is reduced to �1 kcal/mol. In the double mutant(Fig. 2 E), the energy barrier is almost completely elimi-nated. Additionally, the open-state energy well is consid-erably lower (DGTM

opening � �3 kcal/mol) than that of the

TABLE 1 Measurements from PMF Calculations

Construct Interhelix Separation (A)

Conformation Closed Open

WT 16.53 (50.35) 25.51 (52.65) �7.

A18F 16.14 (51.45) 28.18 (51.22) �6.

A25F 17.54 (50.74) 27.06 (50.74) �5.

DM 17.45 (50.70) 30.89 (51.82) �6.

closed-state energy well for the double mutant. This sub-stantial lowering of the open-state energy may reflect addi-tional favorable interactions between the phenylalanineresidues and each other (for, e.g., the simulations showedevidence of p-stacking of the A18F aromatic rings;Fig. S1) or via hydrophobic contacts between the A25Frings and solvating lipid chains.

We caution that direct, quantitative comparison ofthese minimal energies is incomplete if considering therelative probabilities of closed and open states (Boltz-mann-weighted populations). We avoid this calculationhere, as we did in Lewis et al. (35), because we do notknow the appropriate range over which the open

Free Energy (kcal/mol) Energy Barrier (kcal/mol)

Closed Open Closed to Open

92 (51.69) �6.29 (51.39) 3.32 (50.83)

69 (51.45) �7.20 (51.20) 1.38 (50.38)

95 (50.82) �6.21 (50.08) 1.20 (50.81)

21 (51.71) �9.06 (52.03) 0.55 (50.37)


Vunnam et al.

conformations can actually exist (given unknown con-straints in the open state of the full-length receptor). None-theless, these PMF calculations clearly predict that the A18and A25 residues are capable of modulating the closed-to-open conformational dynamics of isolated TM-domaindimers.

TRAIL-induced opening and activation are bothincreased in TM domain mutants

To determine the effects of the simulated mutations on TMconformational dynamics in cellular systems, we introducedthese mutations to DR5LDDD/ECFP/EYFP constructs andperformed TR-FRET. First, we confirmed that there wasno loss of the disulfide-linked dimer population as a resultof the mutagenesis. HEK293 cells were transiently trans-fected with DR5LDDD/ECFP or DR5LDDD/A18F/A25F/ECFP, and whole cell lysates were analyzed under reducing(1 mM DTT) or nonreducing conditions (Fig. S2). Tran-sient expression of the DM had no quantifiable effect onthe population of disulfide-linked dimers as compared toWT (quantified using the software ImageJ; National Insti-tutes of Health, Bethesda, MD). Then, fluorescence life-times were measured 5TRAIL (1 mg/mL). The single and

FIGURE 3 Live-cell TR-FRET studies confirm that point mutations within the

Shown here are spectral decay curves of donor-only (DR5LDDD/CFP) and dono

times (insets) for WT (A), A18F (B), A25F (C), and DM (D). Decrease in lifetim

rophores are close to one another. Figures are representative of four independen


double mutants each showed longer lifetimes than WT inthe presence of TRAIL (Fig. 3, A–D), indicating an increasein the spacing between the fluorophores. Consistent with thePMF predictions, the increase in spacing between the fluo-rophores in the double mutant was significantly greatercompared to WT (FRET efficiency of 5 vs. 16%) than thesingle mutant compared to WT (14 vs. 16%) (Fig. 4 A).Although our main focus is on the role of the TM domainin ligand-induced receptor activation, we were interestedto see how the receptor behaved absent the ligand. As inthe case with ligand, there was no change in ligand-freeFRET efficiency for the single mutants compared to WT(Fig. S3; Table S1). Surprisingly, there was an increase inligand-free FRET for the double mutant (Fig. S3), whichwe cannot currently explain but which may relate to a re-packing of the TM helices that is irrelevant to activation.

We then evaluated if there is a correlation between theligand-induced TM opening in the mutants and receptorsignaling activity. DR5-deficient BJAB cells were tran-siently transfected with full-length DR5L plasmid (WT,A18F, A25F, and DM), and 24–48 h after transfection celldeath was assessed in the presence (0.1 mg/mL) and absenceof ligand. We used a lower dosage of TRAIL to see whetherTM domain mutations enhanced the sensitivity of DR5 to

TM domain of DR5L increase the TRAIL-induced opening and activation.

r-with-acceptor (DR5LDDD/YFP) pairs in the presence of TRAIL and life-

e is associated with increased resonant energy transfer, implying the fluo-

t experiments. To see this figure in color, go online.

FIGURE 4 A positive correlation was observed through increase in distance between the intracellular portions of TRAIL-bound DR5L and apoptotic ac-

tivity of the receptor. (A) Given here are FRET efficiencies for WTwith and without ligand (columns 1 and 2) and for the mutants with ligand at a saturated

concentration of 1 mg/mL (columns 3–5). (B) Given here is the percent cell death calculated from the results of an MTT assay for controls ((C) CþT), WT,

and mutants. TRAIL concentration was 0.1 mg/mL. (C) Given here is the percent cell death versus FRET efficiency. The fit gives an r2 of 0.94. To see this

figure in color, go online.


ligand. To ensure a quantitatively meaningful comparisonbetween constructs, we first determined the cell surfaceexpression levels of WT and the mutants by surface stainingwith phycoerythrin-conjugated mouse monoclonal anti-human DR5 antibody (Anti-Human CD262 (DR5) PE)(Fig. S4 A). Mutants showed 5–10% less surface expressionthan WT (Fig. S4 B), ensuring that any increase in mutantactivity is not due to increased receptor number. Next, wemeasured the cell viability5TRAIL. Strikingly, in the pres-ence of ligand, the DM showed significant increase inapoptotic activity (40% cell death) over both the WT (15%cell death) and single mutants (23% cell death) (Fig. 4 B).Collectively, these results indicate that the double mutationresulted in a correlated increase in separation betweenthe intracellular portions of TRAIL-bound DR5L and anincreased apoptotic activity of the receptor (Fig. 4 C). In theabsence of ligand,WTand the mutants showed no significantdeath when compared to the cells only (Fig. S4 C). As dis-cussed below, these results suggest that the mutations aloneare not enough to constitutively activate DR5L signaling,suggesting that the loss of the TM domain transition barrierand increased energetic favorability of the open state arenot enough to overcome the energy requirements for openingof the ECD andDD.Additionally, it points to amore complexinterplay of interactions with cytosolic adaptor proteins and,likely, physical properties of the membranes (54–56).

DISCUSSION

The experimental results in the presence of TRAIL (bothFRET and cell death) correlate well with the PMF calcula-tions. There are two relevant aspects of the correlationthat bear mention. First, the energy barrier between theclosed and open states is almost completely eliminated inthe PMF of the double mutant. Second, the energy differ-ence between the two states is inverted (GWT

open >GWTclosed,

whereas GDMopen <GDM

closed). Both of these measures correlate

with the experimental results in the presence of ligand(Fig. 5, A–D). However, it is significant that despite this cor-relation, the TM domain mutants did not increase constitu-tive activity of the receptor in the absence of ligand. Thiseffect could not have been predicted by the PMF calcula-tions, which were on the isolated TM domains and thereforeignore any free energy requirements for the opening of theother two domains of the receptor (the ECD and the DD).We can speculate that the TM dimer is prevented from open-ing because the free energy required to open the entire re-ceptor ðDGTNFR

openingÞ is more positive than that gained by theopening of the TM domain with the double mutant(DGTM-DM

opening � �3 kcal/mol; Table 1).This raises a still unclear but fundamental question: in

the overall energetic landscape of receptor activationðDGTNFR

openingÞ, what is the relative role of the wild-type TMdomain closed-to-open transition (DGTM

opening � 2 kcal/mol)in the context of the rest of the receptor? This is a broadlyimportant question that has remained unanswered acrosssuperfamilies where various TM domain conformationalstates have now been observed (17,19,23,57,58). To guidediscussion of this point, there is growing support for a newmodel of TNFR activation, in which preassembled dimersbind ligand and nucleate the formation of multimeric com-plexes. Recent superresolution microscopy data suggestssmall ligand-receptor complexes consisting of 3–6 receptors(Fig. 6) (59). Consistent with the data presented here, thismodel proposes that the free energy of ligand binding isharnessed to force the central dimeric signaling unit into anopen conformation that initiates downstream signaling.There is strong evidence that the dimer is the relevantsignaling unit, including the presence of dimeric complexesin the signaling cascade (8,60). This includes a dimeric deathdomain of the p75 neurotrophin receptor, which, based onfluorescence anisotropy measurements, was estimated tohave a binding free energy of��6 kcal/mol (61). This bind-ing free energy can be used as a rough estimate to


FIGURE 5 Live-cell TR-FRET and cell viability results correlate well with the PMF calculations. (A) Shown here is the FRET efficiency versus the free

energy change associated with the PMF calculations as the protein traverses the reaction coordinate from a closed to a more separated conformation. As free

energy change become less favorable (more positive), FRET efficiencies increase, indicating the DR5L-DR5L interactions are at a closer, less separated

range. R2 for this plot is 0.75. (B) Given here is a correlation between percent cell death from cell viability assay and the free energy change associated

with the conformational separation as predicted from the PMF calculations. As the open conformation becomes less energetically favorable (more positive),

the percent cell death decreases, reinforcing our prediction that the population distribution favoring open conformation is associated with more cell death.

R2 for this is 0.85. (C) Given here is the FRET efficiency versus the height of the free energy barriers between closed and open states. The fit gives an r2 of

0.74. (D) Given here is the percent cell death versus the height of the free energy barriers between closed and open states. The fit gives an r2 of 0.77. To see

this figure in color, go online.

Vunnam et al.

contextualize the free energy required to open the dimericDD of p75 neurotrophin receptor and other TNFRsðDGDD

openingÞ.With regard to the ECD, there is no previous thermody-

namic data published to suggest the free energy require-ments for conformational changes in this domain. Wepreviously used normal mode analysis to predict theintrinsic motions of the TNFR1 ECD, and showed that incombination, the lowest normal modes predict an outwardopening, consistent with the motion of the connected TMdomain dimer (35). Here, to further explore the possible en-ergetic requirements of the ECD, we calculated, to ourknowledge, a new PMF for the opening of the ECD.Fig. 7 shows a predicted �6 kcal/mol ðDGECD

openingÞ energy


barrier to ECD opening. Using the using the ABF approach(49–51) means that the PMF traces the likely lowest-free-energy conformational change pathway from the closed tothe open state.

There are, of course, caveats to a stitched-together (62)total free energy of opening for TNFR dimers, DGTNFR

opening ¼DGECD

opening þ DGTMopening þ DGDD

opening � 14 kcal/mol. Princi-pally, we are culling information from three separate recep-tors, namely TNFR1 (ECD), DR5L (TM), and p75 (DD),which are single-pass, type-1 transmembrane receptorsbelonging to the TNFR superfamily. They share commonstructural features: a cysteine-rich extracellular domain,which functions as a ligand binding domain, a transmembranedomain, and a cytoplasmic death domain. Sequence

FIGURE 6 Proposed mechanism of DR5L activation. This model suggests that activation of the TNF receptor requires opening of the TM domain, which

needs binding of two ligands. To see this figure in color, go online.


alignment DR5L showed 50 and 49% similarity with theTNFR1 and p75, respectively. Thus, it is reasonable to gener-alize a rough picture of activation from the distinct receptors.Although we do not intend to claim quantitative rigor to thisestimate, it serves as an interesting launch point to discussthe thermodynamic contribution of the TM domain to recep-tor activation. The free energy estimate of 14 kcal/mol is inter-esting in at least two ways. First, it gives context to the likelyrelative contribution of the TM domain opening transition(�15%) to the overall thermodynamics of receptor activation.This perhaps helps explain why we do not see constitutive ac-tivity in the doublemutant: assuming that the doublemutationin the TM domain causes no changes in the ECD or DD, wecan estimate that the total free energy change for openingthe double mutant (DGDM

opening � 10 kcal/mol) is still prohibi-tively unfavorable for constitutive opening. Nonetheless, ab-sent other factors, a 4 kcal/mol shift in free energy shouldbe observable in the activation (but it is not; see Fig. S4 C).

FIGURE 7 PMF calculation on separation of CRD4 in the TNFR1 ECD,

showing the free energy landscape along the CRD4 separation reaction co-

ordinate.

A study from 2012 reported binding affinities of a seriesof TNF ligands and associated receptors. The best availableand relevant data is from surface plasmon resonance methodin which trimeric ligand binds to monomeric TNFRs (63). Inthe case of DR5L, TRAIL binding nets �10 kcal/mol (64).In the case of TNFR1, TNF-a binding nets �12 kcal/moland LT-a nets �10 kcal/mol (63). This is suggestive that,given our prediction of �14 kcal/mol, a single ligand bind-ing (Fig. 6) event would represent the lower limit of theneeded energy to induce a conformational change, whereasa second binding event (Fig. 6) would seem to provide morethan enough energy. It is still not clear whether one ligand isnecessary, or two, but there are clues from the availablecrystal structures, specifically that the first structures wereof a single ligand binding three receptors, and later of thepreligand dimer. This suggests a likely dimer-of-trimersmodel as the minimal unit (Fig. 1 A), which would alignwith the two-ligand model for dimer activation. In thatcase, there would be roughly 20–24 kcal/mol in bindingenergy, more than enough to eclipse the �14 kcal/mol sug-gested here.

Despite these estimates, we are still left to discuss whythere is no effect of the mutants on ligand-free activation.To this point, we and others have shown that ligand-induced DR5 activation requires membrane cholesterol(33,65) and, relatedly, is associated with translocationinto lipid rafts (53,66). Bocharov et al. (56) have pointedto the importance to receptor activation of 1) membranethickness and 2) membrane fluidity and elasticity moduli,both of which are altered in lipid rafts. We previouslyshowed that membrane cholesterol increased the propen-sity of the TM domains to form disulfide bonds (6,33).Additionally, interactions with downstream signaling


Vunnam et al.

partners depend on lipid raft partitioning (67–69). Themolecular effects of death domain opening have yet tobe determined, but we can speculate that this processexposes binding surfaces for the cytosolic machinery (inthe case of DR5, the so-called death-inducing signalingcomplex). Thus, even if the nonligand-bound doublemutant were to be open, it would not signal because thereceptor is not in the appropriate membrane environment.Exactly what molecular mechanisms drive the ligand-induced colocalization of DR5 with cholesterol-rich do-mains remains to be seen.

Regardless of these interesting, but unresolved, detailsregarding the energetics of TNFR activation, our studyprovides, to our knowledge, the first clear demonstrationthat the TM domain is influential enough to alter the effi-ciency of the receptors. This has important implicationsfor ongoing efforts to develop TNFR inhibitors, all of whichcurrently target the ECD (70). There are several recentstudies in other receptor superfamilies showing that TMdomains can be powerful targets (e.g., TM-domain-like pep-tides that interfere with ERB2 (71), MUC1-C oncoprotein(72), and the G protein-coupled receptors (73)). The datapresented here suggest that the TNFR TM domain is alsoa promising target for therapeutic intervention.

SUPPORTING MATERIAL

Four figures and one table are available at http://www.biophysj.org/

biophysj/supplemental/S0006-3495(17)30598-2.

AUTHOR CONTRIBUTIONS

N.V. performed all the experiments. C.K.C.-B. conducted and analyzed the

simulations on DR5L and contributed to some FRET experiments. A.K.L.

conducted and analyzed the simulations on TNFR1. J.N.S. provided input

for data interpretation. J.N.S., N.V., and C.K.C.-B wrote the manuscript.

ACKNOWLEDGMENTS

We thank James Gumbart and Hyea Hwang for valuable discussions. Time-

resolved FRET studies were performed at Fluorescence Innovations, Inc., at

the University of Minnesota. We thank Benjamin D. Grant from Fluores-

cence Innovations, Inc., for technical support. This work was carried out

in part using computing resources at the University of Minnesota Super-

computing Institute.

This work was supported by US National Institutes of Health R01

GM107175.

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Biophysical Journal, Volume 113

Supplemental Information

Death Receptor 5 Activation Is Energetically Coupled to Opening of the

Transmembrane Domain Dimer

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

Supplementary Information

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

Nagamani Vunnam1#, Cecily Kristine Campbell-Bezat1#, Andrew K. Lewis1 and Jonathan N. Sachs1*

# These authors contributed equally to this work

*Corresponding Author

1Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455

†This work is supported by National Institutes of Health Grant R01 GM107175

* Department of Biomedical Engineering

University of Minnesota

Twin-cities, MN 55455

Phone: 612-624-7158

Email: [email protected]

KEY WORDS

Tumor necrosis factor receptors

Death receptor 5

TNF- related apoptosis inducing ligand

Disulfide-linked transmembrane dimer

Time-resolved FRET

Supplementary Fig 1

Supplementary Fig. 1. Simulations showed evidence of π- π stacking of the A18F aromatic rings. Rendering illustrating evidence of π- π stacking of the A18F aromatic rings. The red are from A18F and blue rings are from double mutant.

Supplementary Fig 2

Supplementary Fig. 2. Double mutation had no effect on disulfide-linked dimerization of DR5L. Effect of double mutation on disulfide-linked dimerization was determined by immunoblotting. Whole cell lysates of wild type and the double mutant were analyzed by electrophoresis, followed by western blotting with antibody against DR5L. Figure is representative of two independent experiments.

WT WT DM DM

DTT - + - +

β-Actin

Dimer

Supplementary Fig 3

Supplementary Fig. 3. DR5L and the mutants exist as oligomers in the absence of ligand. FRET efficiency of WT and the mutants in the absence of TRAIL. Results are mean ± SD of triplicate determinations. Supplementary Table 1. FRET efficiencies of WT and the mutants in the absence of ligand.

FRET efficiencies for WT and the mutants (No ligand)

Constructs FRETEfficiency%Wild-type 25±1A18F 22±2A25F 24±5

A18F/A25F 40±1Functional Assay without ligand

0

10

20

30

40

50

% F

RE

T E

ffic

ienc

y

Wild-type A18F A25F A18F/A25F

020406080100

FRET efficiencies for WT and the mutants (No ligand)

Constructs FRETEfficiency%Wild-type 25±1A18F 22±2A25F 24±5

A18F/A25F 40±1Functional Assay without ligand

0

10

20

30

40

50

% F

RE

T E

ffic

ienc

y

Wild-type A18F A25F A18F/A25F

020406080100

Supplementary Fig 4 A

B

C

Supplementary Fig. 4. WT and the mutants showed no significant death in the absence of ligand. (A) Fluorescence intensity curves of Phycoerythrin-conjugated Mouse Monoclonal anti-human DR5 emission with inset detailing differences between WT and mutants. (B) Relative surface expression as obtained in A from transiently transfected cells. (C) Cell viability assay in the absence of TRAIL. DR5L deficient BJAB cells were transfected with wild type and the mutants. After 24-48 hours of transfection, the percentage of cell survival was determined by MTT assay.

FIGURE S1: (A) Fluorescence intensity curves of Phycoerythrin-conjugated Mouse Monoclonal anti-human DR5 emission with inset detailing differences between WT and DM. (B) Relative surface expression as obtained in A from transiently transfected HEK293 cells. (C) Western blot showing relative expression amounts of the two splice isoforms of DR5L (parenthesis). Arrowhead is a non-specific protein in non-transfected cells. (D) Western blot of whole cell lysate and CoIP demonstrates the intact TRAIL binding capacity and dimer formation in the A18F/A18F transmembrane construct. Both dimer (D, arrowhead) and monomer (M, arrowhead) are visible for the full length and fluorescent constructs. (E) Western blot confirms that dimer formation is not notably changed in the double mutant.

Wavelength, nm475 550 625 700

Fluo

resc

ence

Inte

nsity

# 104

0

1

2

3

4Wild TypeA18FA25FA18F/A25F

Pull down Assay with FLAG-Tag Beads DR5/Full Length DR5/Truncated/CFP/YFP

250-

150-

100- 75-

50-

37-

25- 20-

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

TRAIL

D"

M"

Full Length Truncated D DR5/Full Length

Western Blot with +/- DTT

250-

150-

100- 75-

50-

37-

25- 20-

WT WT DM DM DTT - + - +

WT WT DM DM - + - +

β-Actin

DR5/Truncated CFP/YFP

D"

M"

Full Length Truncated E A18F) A25F) DM) Cells)Only)WT)

A B

WT A18F A25F DM

Fluo

resc

ence

Inte

nsity

(x 1

04 )

0

1

2

3

4

C

Wavelength, nm

Fluo

resc

ence

Inte

nsity

# 104

1

2

3

Wild TypeA18FA25FA18F/A25FX: 574.1

Y: 2.962e+04



Fluo

resc

ence

Inte

nsity

# 104

0

1

2

3



250-

150-

100- 75-

50-

37-

25- 20-

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

TRAIL

D"

M"



250-

150-

100- 75-

50-

37-

25- 20-


WT WT DM DM - + - +

β-Actin


D"

M"


A B

WT A18F A25F DM

Fluo

resc

ence

Inte

nsity

(x 1

04 )

0

1

2

3

4

C

Wavelength, nm

Fluo

resc

ence

Inte

nsity

# 104

1

2

3


Y: 2.962e+04

Supplementary Fig 4 A

B

C

Supplementary Fig. 4. WT and the mutants showed no significant death in the absence of ligand. (A) Fluorescence intensity curves of Phycoerythrin-conjugated Mouse Monoclonal anti-human DR5 emission with inset detailing differences between WT and mutants. (B) Relative surface expression as obtained in A from transiently transfected cells. (C) Cell viability assay in the absence of TRAIL. DR5L deficient BJAB cells were transfected with wild type and the mutants. After 24-48 hours of transfection, the percentage of cell survival was determined by MTT assay.



Fluo

resc

ence

Inte

nsity

# 104

0

1

2

3



250-

150-

100- 75-

50-

37-

25- 20-

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

TRAIL

D"

M"



250-

150-

100- 75-

50-

37-

25- 20-


WT WT DM DM - + - +

β-Actin


D"

M"


A B

WT A18F A25F DM

Fluo

resc

ence

Inte

nsity

(x 1

04 )

0

1

2

3

4

C

Wavelength, nm

Fluo

resc

ence

Inte

nsity

# 104

1

2

3


Y: 2.962e+04



Fluo

resc

ence

Inte

nsity

# 104

0

1

2

3



250-

150-

100- 75-

50-

37-

25- 20-

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

WT Lysate

WT After CoIP

DM Lysate

DM After CoIP

TRAIL

D"

M"



250-

150-

100- 75-

50-

37-

25- 20-


WT WT DM DM - + - +

β-Actin


D"

M"


A B

WT A18F A25F DM

Fluo

resc

ence

Inte

nsity

(x 1

04 )

0

1

2

3

4

C

Wavelength, nm

Fluo

resc

ence

Inte

nsity

# 104

1

2

3


Y: 2.962e+04

020406080100

Cel

l Via

bilit

y (%

)

Date post:	30-Jul-2020
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