Biophysical Journal Volume 106 March 2014 L21–L24 L21

Open and Closed Conformations of the Isolated Transmembrane Domainof Death Receptor 5 Support a New Model of Activation

Andrew K. Lewis,† Zachary M. James,‡ Jesse E. McCaffrey,‡ Anthony R. Braun,† Christine B. Karim,‡

David D. Thomas,‡ and Jonathan N. Sachs†*†Department of Biomedical Engineering and ‡Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota,Minneapolis, Minnesota

ABSTRACT It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurallyhomologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptormonomers. We and others have proposed an alternate model in which the TNF-receptor dimer—sitting at the vertices of alarge supramolecular receptor network of ligand-bound receptor trimers—undergoes a closed-to-open transition, propagatedthrough a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combinedelectron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of thelong isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic tothe physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.

Received for publication 28 October 2013 and in final form 29 January 2014.

*Correspondence: jnsachs@umn.edu

Editor: Scott Feller.

� 2014 by the Biophysical Society

http://dx.doi.org/10.1016/j.bpj.2014.01.044

Death receptor 5 (DR5) is a member of the tumor necrosisfactor receptor (TNFR) superfamily that mediates apoptosiswhen bound by its cognate ligand, TNF-related apoptosis-inducing ligand (1). Upregulated in cancer cells, DR5 isamong the most actively pursued anticancer targets (2).TNF-related apoptosis-inducing ligand binds to preassem-bled DR5 trimers at their extracellular domains, causingthe formation of oligomeric ligand-receptor networks thatare held together by receptor dimers (3). In the long-isoformof DR5, this dimer is crosslinked via ligand-induced disul-fide bond formation between two transmembrane (TM)domain a-helices at Cys-209, and is further stabilized by aGxxxG motif one helix-turn downstream (3).

Our recent study of the structurally homologous TNFR1showed that receptor activation involves a conformationalchange that propagates from the extracellular domain tothe cytosolic domain through a separation (or opening) ofthe TM domains of the dimer (4). We have therefore hypoth-esized that the activation of DR5, and indeed all structurallyhomologous TNF-receptors, involves a scissorslike openingof the TM domain dimer (Fig. 1).

Using electron paramagnetic resonance (EPR) spectros-copy, a technique that has been used previously to studyTM helix architecture and dynamics (5,6), and potential-of-mean force (PMF) calculations (7,8), this study addressesthe question of whether the isolated disulfide-linked DR5-LTM domain dimer occupies distinct open and closed states(Fig. 1), and how its dynamic behavior contributes to thefree-energy landscape of the opening transition of the full-length receptor.

The DR5-L TM domain was synthesized with TOAC,an amino acid with a nitroxide spin label rigidly fixed tothe a-carbon (9), incorporated at position 32 (Fig. 1), with

some minor modification to facilitate EPR measurements.Previous work confirmed that this peptide forms disulfide-linked dimers (e.g., via comparison to 2-ME treated sample)and a negligible population of higher-order oligomers(further supported by model fitting of the EPR data below).For peptide work, residues were renumbered such that Thr-204 corresponds to Thr-1, and so on. The cytosolic Cys-29(which we previously showed does not participate in adisulfide bond in cells) was replaced with serine to preventthe formation of antiparallel disulfide-linked dimers, andTrp-34 was replaced with tyrosine to prevent intrinsic fluo-rescence in fluorescence studies (not published). Contin-uous-wave (CW) dipolar EPR (sensitive only to spin-spindistances <25 A) was used to measure TOAC-TOACdistances within the TM dimers and revealed an orderedGaussian distribution centered at 16 A (full width half-maximum (FWHM) ¼ 4 A), corresponding to a closed state(Fig. 2 A). Double electron-electron resonance (DEER)(sensitive to spin-spin distances from 15 to 60 A) also de-tected a short distance consistent with the dipolar EPRdata, along with a longer, disordered component (32.9 A,FWHM ¼ 28 A) (Fig. 2 B). Together, these measurementsindicate the presence of a compact, ordered closed stateand a broader, disordered open state. EPR on oriented mem-branes also indicated two structural states. Global fitting re-vealed two populations of spin-label tilt angles (orientationof the nitroxide principal axis relative to the membranenormal): a narrow conformation (24�, FWHM ¼ 20�), and

FIGURE 1 Activation model of the DR5-L TM dimer. The

sequence and positions of the disulfide bond and TOAC spin

label (top), along with our previously published model (bottom,

left) are shown. We propose an activation model (bottom, right)

in which the transmembrane dimer pivots at its disulfide bond to

reach an active open conformation.

FIGURE 2 EPR spectra (left) of 32-TOAC-DR5 in lipid, and

resulting structural distributions (right). (A) CW dipolar EPR

spectra (left) of dimer (1 mM diamide) and monomer (1 mM

2-mercaptoethanol). Best-fit spin-spin distance distribution

was a single Gaussian centered at 16 5 2 A (right). (B) The

DEER waveform (left) of 32-TOAC-DR5 dimer was best fit (right)

to a two-Gaussian distribution. The short distance was con-

strained to agree with the CW data, because DEER has poor

sensitivity for distances <20 A. The long-distance distribution

is centered at 32.9 A and is much broader. (C) CW EPR spectra

(left) of 32-TOAC-DR5, with the membrane-normal oriented par-

allel (red) and perpendicular (blue) to the field. Simultaneous

(global) fitting of these spectra reveals narrow and broad com-

ponents (right). (In panels B and C, the overall distribution is

plotted as black, while the closed and open components are

plotted as green and magenta, respectively.)

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a disordered conformation (50�, FWHM ¼ 48�) (Fig. 2 C).This bimodal orientational distribution (Fig. 2 C) is remark-ably consistent with the bimodal distance distribution(Fig. 2 B).

We subsequently conducted a PMF calculation (10) usingthe DR5-L TM dimer starting configuration developed byour group previously (3), embedded in a DMPC bilayer,with the Leu-32/Leu-32 Ca distance as the reaction coordi-nate. Three calculations were run from independent startingconfigurations, each using 50 windows spaced in 0.5� incre-ments, and run for 20 ns at each window (totaling 3 ms).Each of the calculations yielded a similar result, and theaveraged free energy curve (Fig. 3 A) agrees remarkablywell with our EPR measurements: a narrow distributionat the closed conformation (~16 A, Fig. 3 B) separatedby an ~3 kcal/mol energy barrier from a broad distributionof accessible open conformations at ~27 A, (Fig. 3 C).Each of the three individual PMF plots can be found inFig. S1 in the Supporting Material.

In the closed state, the helices are tightly packed at theGxxxG interfacial motif and all the way down the juxta-posed helix faces at residues Ala-18, Leu-22, Ala-25, andVal-26. The tight packing is aided by kinking and twistingof the two helices around their common axis, increasingthe interacting surface area. In the open conformations,the Ala-18, Leu-22, Ala-25, and Val-26 pairs are dissoci-ated and, interestingly, the GxxxG motif at Gly-10 andGly-14 remains tightly packed. The open state energywell is only slightly less favorable than the closed state(by ~2 kcal/mol), and its free energy profile is relativelybroad and flat. The increased crossing angle in the openstate is facilitated by straightening of the helix kink andis not accommodated by a change in bilayer thickness(see Fig. S3, A and B).

The observed change in helix-helix distance (11 A be-tween the two minima in the PMF) is extremely close tothat observed previously in live-cell FRET studies of aconstitutively active form of TNFR1 (~8 A change betweenstates using large fluorescence probes at the cytosolicdomains) (4). The change observed in the EPR data

Biophysical Journal 106(6) L21–L24

(17 A) may be an overestimate because the measurementis made between TOAC spin labels that likely protrudefrom the two helices, depending on rotational orientation.These results collectively show that activation of thesereceptors requires a small, but clearly significant conforma-tional opening of the TM domains. One important note isthat our EPR experiments recapitulate the equilibrium dis-tribution of the two states despite there being no drivingforce to traverse the barrier between them (~3 kcal/molin the closed-to-open transition and ~1 kcal/mol in theopen-to-closed transition, Fig. 3). We do not interpret theresults to mean that the dimer necessarily traverses thesebarriers at 4�C. Rather, there likely exist multiple reactionpaths for dimerization of the abstracted TM domains.Finally, in the context of the full-length receptor, how

FIGURE 3 (A) PMF calculation of the DR5 TM domain dimer

along the Leu-32/Leu-32 distance reaction coordinate. The

PMF calculation reveals a narrow closed state and a broader

open state separated by a free energy barrier. Representative

snapshots of the (B) closed state and (C) open state.

Biophysical Letter L23

the ligand induces a conformational change capable of over-coming the closed-to-open barrier remains an importantquestion.

Whether the observed structural transition in the TMdomain dimer of the long-isoform of DR5 is a ubiquitousconformational switch that acts over the entire TNFR super-family remains unknown. Vilar et al. (11) first proposed asimilar scissors-model for activation of p75 neurotrophin re-ceptor, which has a cysteine at the center of its TM helix.The short isoform of DR5 lacks a TM domain cysteine,but does form noncovalent dimers in cells, with likely TMdomain dimer contacts (3). Among the other closely relatedand structurally homologous members of the TNFR super-family, TNFR1 contains a cysteine at the center of the TMdomain, but lacks any discernible small residue motifs(e.g., GxxxG). TNFR2 lacks a TM cysteine on the extracel-lular side, but does have a GxxxG motif positioned similarlyto that of DR5. On the other hand, Death Receptor 4, whosefunctional distinction from DR5 has remained somewhat

elusive, lacks both a cysteine and any recognizable small-residue hydrophobic motif.

In summary, we have extended recent findings that pointto the TM domain of DR5 as an essential structural compo-nent in the conformational change associated with activa-tion. Our findings that the DR5-L TM domain occupiesdistinct open and closed states, separated by a substantialenergy barrier, points the way to further studies across theTNF-receptor superfamily.

SUPPORTING MATERIAL

Three figures along with Methods section, with EPR Sample Preparation,

EPR Spectroscopy, PMF Calculation and Molecular Dynamics and

references (12–19) is available at http://www.biophysj.org/biophysj/

supplemental/S0006-3495(14)00176-3.

ACKNOWLEDGMENTS

Simulations were carried out using resources at the University of Minnesota

Supercomputing Institute. Electron paramagnetic resonance experiments

were performed at the Biophysical Spectroscopy Center, University of Min-

nesota. D.D.T. was supported by the National Institutes of Health grant No.

GM27906.

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