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Letter
Nature Structural Biology 6, 1048 - 1053 (1999)
doi:10.1038/14935
¤2¤ Structure of the TRAIL−DR5 complex reveals mechanisms conferring
specificity in apoptotic initiation ¤2¤
Juthathip Mongkolsapaya^1, Jonathan M. Grimes^2, Nan Chen^1, Xiao-Ning
Xu^1, David I. Stuart^2, ^3, E.Yvonne Jones^2, ^3 & Gavin R.
Screaton^1, ^4
^1 MRC Human Immunology Unit, Institute of Molecular Medicine, John
Radcliffe Hospital, Oxford OX3 9DS, UK.
^2 Structural Biology, Wellcome Trust Centre for Human Genetics,
Roosevelt Drive, Headington, Oxford, OX3 7BN, UK.
^3 Oxford Centre for Molecular Sciences, New Chemistry Building, South
Parks Road, Oxford, OX1 3QT, UK.
^4 Nuffield Department of Medicine, John Radcliffe Hospital, Oxford,
OX3 9DU, UK.
Correspondence should be addressed to E.Yvonne Jones
[59]Yvonne.Jones@strubi.ox.ac.uk
TRAIL, an apoptosis inducing ligand, has at least four cell surface
receptors including the death receptor DR5. Here we report the crystal
structure at 2.2 resolution of a complex between TRAIL and the
extracellular region of DR5. TRAIL forms a central homotrimer around
which three DR5 molecules bind. Radical differences in the surface
charge of the ligand, together with variation in the alignment of the
two receptor domains confer specificity between members of these ligand
and receptor families. The existence of a switch mechanism allowing
variation in receptor domain alignment may mean that it is possible to
engineer receptors with multiple specificities by exploiting contact
positions unique to individual receptor−ligand pairs.
EST database searching has led to the cloning and characterization of a
number of proteins belonging to the tumor necrosis factor (TNF) and TNF
receptor (TNF−R) families. Although structurally related, members of
the TNF−R family subserve a number of different functions such as
activation, cell growth and differentiation in addition to apoptosis. A
subset of the receptors (TNF-R1, Fas, DR3, DR4, DR5 and DR6) possess an
intracellular death domain that can initiate a series of
protein−protein interactions leading to cell death^[60]1, ^[61]2.
TNF related apoptosis inducing ligand (TRAIL) was identified by
database searching and shown to kill a variety of tumor cell lines
while showing little activity against normal cells^[62]3. The
widespread expression of TRAIL (shown by Northern blotting)^[63]3, and
the lack of sensitivity of most cell types to its effects, initially
suggested that the expression of the TRAIL receptors would be tightly
controlled. However when the two TRAIL receptors DR4 and DR5
(TRICK2/TRAIL-R2/Killer) were cloned they were also found to be widely
expressed on many cell types, most of which were insensitive to the
effects of TRAIL^[64]2. Two further TRAIL receptors have been
identified: decoy receptor 1 (DcR1) (LIT/TRAIL-R3/TRID) and DcR2
(TRAIL-R4/TRUNDD)^[65]2. The four TRAIL receptors possess two cysteine
rich repeats, in contrast to TNF-R1 and TNF-R2, which each have four,
and Fas, which has three. DR4 and DR5 have death domains, while DcR1
lacks a cytoplasmic domain and DcR2 contains an incomplete
non-functional death domain. DcR1 and DcR2 are therefore thought to act
as decoys, protecting cells that coexpress either DR4 or DR5 from
apoptosis induced by TRAIL. Thus, following activation, peripheral
blood lymphocytes that are initially insensitive to TRAIL lose
expression of DcR1 and subsequently become sensitive to TRAIL^[66]4.
The differential sensitivity of tumors compared to normal cells
suggested that TRAIL may have greater utility as an anti-tumor agent
than TNF, which has limited use because of its side effects. In a
murine model, TRAIL has been shown to be effective against xenogenic
tumor transplantation without harming the host^[67]5.
The emerging complexity of the TNF and TNF-R families raises questions
at the molecular level, such as what is the basis of specificity and
more particularly for the TRAIL−TRAIL receptor system, what are the
conserved features that confer the ability to initiate apoptotic
signaling. We have determined the crystal structure of the TRAIL−DR5
complex, which throws light on these questions, revealing an unusual
mechanism for controlling specificity.
Structure determination
The complex between soluble forms of recombinant human TRAIL (residues
91−281, all numbering starts from the initiation codon) and DR5
(residues 58−184) crystallized with an asymmetric unit consisting of
one subunit of TRAIL and one copy of DR5, forming the full trimeric
complex through crystallographic three-fold symmetry. The crystals
diffracted to 2.2 using synchrotron radiation and the structure was
phased by a combination of molecular replacement and heavy atom
derivatives. With the exception of some mobile loop regions, all of
TRAIL C-terminal to residue 119 and the cysteine rich repeat region of
DR5 (starting at residue 69) are well ordered in the final crystal
structure (crystallographic R factor 22.1% and R[free] 27.0%; see
Methods and [68]Table 1). The overall structure of the TRAIL−DR5
complex, including representative electron density, is shown in
[69]Fig. 1.
[70]Figure 1. The structure of TRAIL, DR5 and the TRAIL−DR5 complex.
[71]Figure 1 thumbnail
a, Stereo view of the complex. The three crystallographically
equivalent copies of the TRAIL subunit (yellow, cyan, pink) and DR5
(blue, green, red) are depicted schematically and the TRAIL trimer is
enclosed in a transparent molecular envelope. This orientation defines
a standard view. b, The complex as depicted in (a) but viewed down the
three-fold axis. The orientation is such that the cell surface
presenting DR5 is above the page and that for TRAIL is below the page.
c, Superposition of TRAIL (pink) with TNF beta (blue). The secondary
structure elements for TRAIL are also marked on the sequence alignment
in [72]Fig. 2a. The r.m.s. deviation is 0.9 for 120 structurally
equivalent C alpha atoms. The major extension of the AA" loop in TRAIL
is highlighted by yellow stripes. The cell surface position is not to
scale. d, Comparison of DR5 and TNF-R1. DR5 and TNF-R1 (from the TNF
beta −TNF-R1 complex) are depicted schematically in the left and right
panels respectively with equivalent regions in identical colors. The
central panel is based on superposition of DR5 D1 and TNF-R1 D2. The
schematic representation of TNF-R1 is shown in gray while that of DR5
is in green. Disulfide bonds are depicted in yellow as ball-and-stick
representation. e, Portion of the final 2F[o] - F[c] electron density
map contoured at 1 sigma , showing a portion of the TRAIL structure in
the BC loop.
[73]Full Figure [74]Full Figure and legend (68K)
[75]Table 1. Crystallographic Statistics
[76]Table 1 thumbnail
[77]Full Table [78]Full Table
The TRAIL trimer
The TRAIL subunits consist of beta -sandwiches conforming, as expected,
to the jellyroll topology^[79]6. Each TRAIL monomer contains one
cysteine at position 230. Crystallization was performed under
non-reducing conditions and in the crystal a disulfide bond was seen
between two of the three cysteine residues in each trimer. The
formation of this disulfide bridge did not affect receptor binding, as
both TRAIL that had been refolded in iodoacetamide and a TRAIL mutant
(C230S) immunoprecipitated DR5 in a manner similar to that of wild type
TRAIL (data not shown). The trimeric interface is extensive with a
large population of aromatic residues conferring a highly hydrophobic
character (66% of the 2,250 ^2 buried surface per subunit is apolar
([80]Fig. 2a). Comparison with the other known structures for members
of the TNF superfamily (TNF alpha , TNF beta /LT alpha and
CD40L)^[81]6, ^[82]7, ^[83]8 highlights a high level of structural
conservation within the beta -strands and at the trimeric interface
([84]Figs 1c, [85]2a), despite the relatively low sequence identity
between the family members (23−36% for the sequences in [86]Fig. 2a).
Of particular interest are strands D and E, which have highly divergent
sequences, yet are almost completely superimposed in structural
alignments. Structural variation in the family is concentrated in the
loop regions, with the most dramatic differences seen in the AA", CD
and EF loops. The TRAIL structure is unique within the TNF family in
displaying a major extension of the AA" loop that is 16 amino acids
longer than in TNF beta ([87]Fig. 1c). In the final stages of preparing
this manuscript the 2.8 resolution crystal structure of uncomplexed
TRAIL was reported^[88]9. In the absence of coordinate deposition we
have not been able to make a detailed comparison of the liganded and
unliganded structures of TRAIL, however inspection of the figures
suggests that the N-terminal portion of the AA" loop has a markedly
different conformation in the two structures.
[89]Figure 2. Comparison of the TRAIL−DR5 and TNF beta −TNF-R1
complexes.
[90]Figure 2 thumbnail
a, Sequence alignments for the TNF and TNF-R1 families. Residues in
TRAIL, TNF beta , DR5 and TNF-R1 are color coded according to their
contribution to the interaction patches in (b). Sequence alignments to
TRAIL are based on pairwise structural superpositions for TNF beta ,
TNF alpha and CD40L and on manual sequence comparison for FasL.
Secondary structure assignments and dots at every tenth residue above
the sequences are for TRAIL. Triangles below the sequences indicate
TRAIL residues buried in the isolated molecule (less than 20% relative
side chain solvent accessible area^[91]27), circles indicate surface
exposed residues mutated in TNF alpha and/or TNF beta (reviewed in ref.
[92]30). Sequence alignments to DR5 are based on pairwise structural
superposition for TNF-R1 and on manual sequence comparison for the
remaining molecules. Dots above the sequences mark every tenth DR5
residue. Symbols in rows 1 and 2 below the sequences indicate the
nature of interactions involving DR5 and TNF-R1, respectively. Circles
in row 3 indicate candidate residues for TRAIL binding specificity in
DR5, DR4, DcR1 and DcR2. b, Surface representations of TRAIL, DR5, TNF
beta and TNF-R1. The relative orientation of the two complexes is
defined by pairwise superposition of the TRAIL and TNF beta subunits.
The view of the TRAIL and TNF beta surfaces corresponds to that of
[93]Fig. 1a while the matching DR5 and TNF-R1 surfaces are revealed by
a 180¡ rotation as in the opening of a book. The solvent accessible
surfaces (calculated in isolation) of each component are shown in gray
with areas contributed by residues implicated in the interaction
surface of the complex highlighted in colored patches. Colors for
interaction patches are matched between ligand and receptor for each
complex. Conservation of the color scheme is used to highlight broadly
equivalent regions of interaction in the two complexes.
[94]Full Figure [95]Full Figure and legend (83K)
The DR5 receptor
Members of the TNF-R family are characterized by extracellular repeats
containing three disulfide bridges with a cysteine knot topology^[96]7,
^[97]10. The number of repeats ranges from six in CD30 through four in
TNF-R1 and TNF-R2 to three in Fas and only two in the four TRAIL
receptors. The crystallographically well-ordered portion of the DR5
molecule includes these two repeats (D1 and D2), which form the ligand
binding region in the complex. DR5 starts with an N-terminal cap
containing a partial cysteine knot with a single non-canonical
disulfide ([98]Fig. 1d). This N-terminal cap corresponds to the
C-terminal half of the first repeat (D1) in TNF-R1, while DR5 D1 and D2
correspond to the central two repeats, D2 and D3, of TNF-R1 which form
the binding interface in the TNF beta −TNF-R1 complex. The sequences of
DR5 D1 and D2 differ considerably, with only 25% amino acid identity
compared to 42% between TNF-R1 D2 and D3. This difference is borne out
at the structural level: the root mean square (r.m.s.) deviation is 1.0
between 30 equivalent C alpha atoms in DR5 D1 and D2 compared to 0.8
between 29 equivalent C alpha atoms in TNF-R1 D2 and D3. The two
ligand binding repeats in these and other TNF-receptor like molecules
are joined by a CXC motif (CQC in all the TRAIL receptors and CGC in
TNF-R1) which acts as a flexible articulation point in the isolated
TNF-R1 structure^[99]10. The complexed structures of DR5 and
TNF-R1^[100]7 show a clear difference in the relative orientation of
the two repeats forming their respective ligand binding regions
([101]Fig. 1d). Thus, a tilt and rotation totaling 36¡ about the CXC
pivot point would be required to place DR5 D2 in an orientation similar
to that of TNF-R1 D3.
The TRAIL−DR5 complex
The TRAIL−DR5 complex reflects the trimeric nature of TRAIL, binding
one copy of DR5 in each of the clefts between subunits ([102]Fig. 1).
The surface area of 1,420 ^2 buried on TRAIL by each DR5 is relatively
equally divided between the two subunits. Likewise, the 1,540 ^2 of
surface buried on the DR5 is shared equally between its two repeats
([103]Fig. 2a,b). This contrasts with the TNF beta −TNF-R1
complex^[104]7, where although the receptor makes approximately equal
contacts with the two TNF subunits, there is a major difference in the
contributions of TNF-R1 D2 and D3, with some two thirds of the contact
area residing on D2.
Superposition of the two complexes shows the position of DR5 D1 and its
equivalent in TNF-R1 to be highly conserved, while a change in tilt
between D1 and D2 of DR5 allows the second domain to more closely
follow the TRAIL surface, introducing a number of novel contacts.
A detailed analysis of the receptor−ligand contacts is shown in
[105]Fig. 2b, where areas of contact are related by color and mapped
back to the sequence alignments ([106]Fig. 2a). There are four main
contact patches on TNF beta (apical subunit 1: A1 (blue), basal subunit
1: B1 (cyan), apical subunit 2: A2 (orange), basal subunit 2: B2 (red);
[107]Fig. 2). The contacts on TRAIL duplicate these areas while
containing additional points of contact in the apical portion of
subunit 1 created by the repositioning of D2 (yellow and green). This
repositioning also alters which regions of the receptors are involved
in the conserved patches (A1-blue and A2-orange) on the apical regions
of the ligands. In the most extreme cases (orange and purple patches)
the residue usage shifts from one cysteine repeat to the other with the
A2 (orange) interaction mediated primarily by residues 107−109 in the
N-terminal binding repeat of TNF-R1 but by residues 153−155 in the C
terminal repeat of DR5. The more topographically conservative elements
of the interaction involve DR5 D1, which makes contact with the B1
(cyan) and B2 (red) patches. Although similarly located, the nature of
the interacting residues in the B2 (red) patches from the two complexes
is highly divergent. The hydrophobic interactions of the B1 patch
(cyan), mediated by the surface exposed Tyr 216 in TRAIL (to Leu 114 in
DR5) and Tyr 142 in TNF beta (to Leu 100 in TNF-R1), are the only truly
conserved portions of the interface ([108]Fig. 3a). Mutagenesis of this
Tyr residue in TNF alpha , TNF beta and FasL abolishes receptor
binding^[109]11, ^[110]12, ^[111]13. Much of the remainder of the
interface in both complexes is made up of polar or charged
interactions. The details of these electrostatic interactions differ
completely between the two complexes ([112]Fig. 3b).
[113]Figure 3. Elements of conservation and specificity in
ligand−receptor binding.
[114]Figure 3 thumbnail
a, Conservation in ligand-receptor interactions. Close up of the
interaction involving the B1 (cyan) surfaces in the TRAIL−DR5 and TNF
beta −TNF-R1 complexes centered on the key tyrosine residue (Tyr 216 in
TRAIL). In this and in (c), the polypeptide chains are represented
schematically as in [115]Fig. 1a and the solvent-accessible surfaces of
the receptors (calculated in isolation) are shown as semi-transparent
envelopes. b, Comparison of surface charge between TRAIL, TNF alpha ,
TNF beta and their receptors. Blue denotes positive, and red negative;
electrostatic potential is contoured at plusminus 8.0 kT in program
GRASP^[116]31. The views of ligand and receptor are as in [117]Fig. 2b.
c, Interaction of Arg 149 in the AA" loop of TRAIL with Glu 147 in DR5.
d, BIAcore analysis showing binding of DR5 to wild type TRAIL or the
mutant lacking the AA" loop. e, Immunoprecipitation with DR5-Fc in the
presence of wild type (W) or the slightly smaller AA" TRAIL mutant (M).
Lanes 1 and 2 immunoprecipitated material (IP), lanes 3 and 4 material
left in supernatant (SN) following immunoprecipitation.
[118]Full Figure [119]Full Figure and legend (83K)
Features conferring specificity
Inspection of the four TRAIL binding receptor sequences shows 45−81%
identity in D1 and 59−80% identity in D2. Most of the residues involved
in the TRAIL−DR5 interface show conserved characteristics, underscoring
the role played by these residues in receptor−ligand specificity
([120]Fig. 2a). D2 is implicated as a major focus for TRAIL-binding
specificity, with conservation in this receptor subgroup of residues
such as Glu 147, Glu 151, Arg 154 and Asp 175 (DR5 numbering) that
mediate complimentary electrostatic interactions. One of these
distinctive TRAIL-binding residues (Glu 147) makes a salt bridge to Arg
149 on the extended AA" loop of TRAIL ([121]Fig. 3c); the shorter AA"
loop in TNF beta precludes any such interaction in the TNF beta −TNF-R1
complex. This electrostatic interaction may help establish the correct
docking orientation for DR5 D2 to TRAIL, stabilizing the appropriate
tilt between D1 and D2. The importance of this contact was demonstrated
by mutagenesis where the 19 amino acid sequence in TRAIL
(^135TLSSPNSKNEKALGRKINS^153) was replaced with three amino acids
(^76SSL^78) present in the much shorter AA" loop of TNF beta . Binding
of the mutant TRAIL assessed by BIAcore was reduced by over 95%
([122]Fig. 3d). In a separate experiment, DR5-Fc was used to
immunoprecipitate either the wild type or the AA" loop mutant of TRAIL.
In agreement with the BIAcore analysis, DR5 binding to the mutant TRAIL
was completely abolished ([123]Fig. 3e). The modeling study of Cha et
al.^[124]9 does not appear to replicate the major differences in
receptor docking observed between the crystal structures of TRAIL−DR5
and TNF beta −TNF-R1. However, the findings of the accompanying
mutagenesis study of the AA" loop are in accordance with those reported
here.
Discussion
The TRAIL−DR5 complex highlights the tight interplay between structure
and function for this receptor−ligand family. Both ligand and receptor
are constructed from robust structural motifs with signaling mediated
by the interaction of receptor with the stable trimeric scaffold of the
ligand. This rigid framework allows free variation of the surface
residues in both ligand and receptor conferring the biological
specificity of particular ligand−receptor pairs through numerous
changes in the interacting pairs of residues. In the ligand, this amino
acid diversity is perhaps most extreme in the surface-exposed D and E
strands and flanking loops where very little sequence homology can be
discerned ([125]Fig. 2a). The surface variation is also clearly
illustrated in [126]Fig. 3b where it can be seen that although the
overall surface topology remains intact, the distributions of charge on
the surfaces of TNF alpha , TNF beta , and TRAIL are very different. In
the receptors, the orientation selected at the flexible CXC junction
determines which areas of the C-terminal ligand binding repeat are
involved in the complex. This switch mechanism may mean that it is
possible to engineer receptors, with multiple specificities by
exploiting contact positions unique to individual receptor−ligand
pairs.
Members of the TRAIL binding subgroup of receptors have, like the
chicken death receptor CAR1 (ref. [127]14), been cut down to a minimal
configuration of only two cysteine-rich repeats, with the second (D2)
tightly abutting the three-fold axis in the TRAIL−DR5 complex.
Superposition of the complete, four repeat, TNF-R1 structure^[128]10
onto the TNF beta −TNF-R1 complex^[129]7 reveals the C-terminus of the
TNF-R1 cysteine rich region at a distance from the three-fold axis that
is similar to that reached more directly by the DR5 C-terminus. The
length of the tether between the cell membrane and second repeat of the
TRAIL receptors varies from two amino acids in the shorter of two
alternatively spliced variants of DR5 (ref. [130]15) to 92 in DcR1. The
functional consequences of this heterogeneity have not been explored
and it is not known whether a single TRAIL trimer can form a complex
composed of receptors with greatly differing tethers. Residue 184 of
DR5, which is visible in the complex structure, is at the start of the
transmembrane helix, meaning that the apex of TRAIL will be positioned
only ~6 from the surface of its target cell. The complex structure
indicates that the DR5 transmembrane helices are arrayed 54 apart.
This is strikingly similar to the 52 spacing of binding sites for the
cytosolic domain of TNF-R2 on the trimeric TRAF2 molecule that
initiates intracellular signaling^[131]16. The extracellular
ligand−receptor complex may thus trigger the apoptotic signal by
dictating a precise positioning of the three transmembrane helices and
hence the cytosolic domains.
[132]Top
Methods
Protein expression, refolding, purification and mutagenesis.
An N-terminally truncated version of TRAIL (amino acids 91−281) was
produced in Escherichia coli strain BL21 DE3 pLys S using the T7 driven
vector pET-9C (Novagen). Inclusion bodies were prepared and solubilized
in 8 M urea and refolded using standard conditions^[133]17. Trimeric
TRAIL was then isolated by gel filtration. The extracellular domain of
DR5 (amino acids 58−184) was produced as an immunoglobulin fusion
protein. The cleavage site for type 14 rhino virus 3C protease^[134]18
was introduced between DR5 and the constant region of IgG1. Fusion
protein (produced by transient calcium phosphate transfection of 293T
cells) was isolated using protein A sepharose and DR5 was cleaved from
the beads using a 3C-GST fusion protein (subsequently removed using
glutathione agarose).
PCR mutagenesis was used to replace the AA" loop of TRAIL
(^135TLSSPNSKNEKALGRKINS^153) with three amino acids (^76SLL^78)
present in the much shorter loop of TNF beta . These changes did not
disrupt the folding of TRAIL, which was shown to be trimeric by gel
filtration and also to have similar secondary and tertiary structure by
circular dichroism. Binding of TRAIL was assessed by surface plasmon
resonance on a BIAcore 2000 machine. Briefly, DR5-Fc (or TNF-R1 Fc as a
negative control) was immobilized onto CM5 sensor chips covalently
coated with a murine anti-human Fc specific monoclonal antibody. A 20
mu M solution of wild type or mutant TRAIL (in 50 mmTRIS pH 8, 150 mM
NaCl) was injected for 60 s.
Crystallization.
Purified DR5 and TRAIL trimer (each at 7 mg ml^-1 suspended in 20
mMTris pH8 and 50 mM NaCl) were mixed at 4:1 stoichiometry. Crystals
were grown by vapour diffusion at room temperature using microbridges
from sitting drops containing 1 mu l of protein solution plus 1 mu l of
reservoir solution (25% ethylene glycol, 0.1% n-octyl- beta
-glucoside). The plate-like crystals (typically 0.5 mm x 0.3 mm x 0.1
mm) were shown to contain both TRAIL and DR5 by polyacrylamide gel
electrophoresis. Crystals were flash-cooled to 100 K in gaseous N[2]
(Oxford Cryosystems).
Structure determination.
Statistics are given in [135]Table 1. All X-ray diffraction data were
collected using synchrotron radiation, auto-indexed and integrated and
scaled with the HKL program package^[136]19. The initial 2.4
resolution X-ray diffraction data set was collected using a Mar345
imaging plate detector (18 cm diameter setting) on beamline ID2 (
lambda = 0.997 , collimation 0.2 mm x 0.2 mm) of the European
Synchrotron Radiation Facility (ESRF). The crystals belong to space
group P321 with unit cell dimensions a = b = 95.2 , c = 69.81 ,
consistent with the crystallographic asymmetric unit containing a
single copy of DR5 and one subunit of TRAIL. Molecular replacement was
carried out using the complete trimeric TNF beta −TNF-R1 complex
(Protein Data Bank accession code 1TNR) with 15−3.5 resolution data in
program AmoRe^[137]20. An unambiguous solution positioned the trimeric
DR5−TRAIL complex on the crystallographic three-fold axis (correlation
coefficient for the top solution 0.324, next highest solution 0.290;
correlation coefficients as defined in ref. [138]20). For refinement
TNF-R1 was cut to comprise only D2 and D3. Initially rigid body
refinement was performed (with the ligand and receptor as separate
bodies), followed by positional and B-factor refinement. These
refinements were stable in XPLOR, using a correlation coefficient
target function^[139]21, although not in CNS^[140]22. The final
R[cryst]was 57.4% (R[free] = 57.8%) on data from 20 to 4 . Despite
the disappointing statistics maps calculated using the maximum
likelihood algorithm encoded in CNS showed good, bias free, electron
density in the region of the ligand. Although the electron density for
the receptor was of poor quality there was clear evidence for a change
in the relative orientation of the two domains.
A high resolution native data set (2.2 resolution) and two heavy atom
derivative sets (5 mM lead acetate soaked for 24 h at room temperature
and 4 mM K[2]HgI[4] in 20 mM KI soaked for 24 h at room temperature)
were collected at 100 K on beamline 9.6 at the CLRC Synchrotron
Radiation Source, Daresbury Laboratory (ADSC Quantum 4 CCD detector,
lambda = 0.87 , collimation 0.25 mm times 0.25 mm). Native and
derivative data were scaled using program SCALEIT^[141]23 and the
derivatives were solved by difference Fourier syntheses. Heavy-atom
parameters were refined and combined MIR and phi [calc] phases were
produced using program SHARP^[142]24. Solvent flattened maximum
likelihood 2F[o] - F[c] and F[o] - F[c] electron density maps
calculated on the basis of these combined phases and the high
resolution native data showed good quality electron density for both
ligand and receptor. The DR5 and TRAIL sequences were fitted to the
electron density using the interactive graphics program O^[143]25.
Further refinement (program CNS), alternated with manual rebuilding,
resulted in the current model ([144]Table 1).
Structural analysis.
For residues 133−143 and 195−201 in the AA" and CD loops of TRAIL the
quality of the electron density is poor but sufficient to define the
course of the polypeptide main chain. Cys 230 and Trp 231 show
statistical disorder in the crystal arising from the formation of a
disulfide between two of the three copies of Cys 230 at the TRAIL
three-fold.
Structural superpositions were performed using the program SHP^[145]26
and a 1.4 probe was used to evaluate surface accessibility with the
program NaccesS^[146]27. Molecular contacts were evaluated in program
CONTACTS (R. Esnouf, pers. comm.) using a 4 cutoff for inter-atomic
distance and the implicated residues mapped as contact patches on the
ligand and receptor surfaces using program VOLUMES (R. Esnouf, pers.
comm.). Figures were produced using Bobscript^[147]28 and were rendered
with Raster3D^[148]29.
Coordinates.
Coordinates for the TRAIL−DR5 complex have been deposited with the
Protein Data Bank (accession number [149]1D4V).
[150]Top
Received 1 October 1999; Accepted 6 October 1999
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[240]Top
Acknowledgments
We thank R. Esnouf, P. Gouet and R. Bryan for computing facilities and
programs, K. Harlos for help with heavy atom soaks, G. Gao for advice
on refolding protocols and crystallisation trials, K. Hudson and J.
Heath for the rhinovirus 3c protease methodology, M. Gross for circular
dichroism analysis, A. van der Merwe and A. McMichael for discussion.
We also thank the staff at the European Molecular Biology Laboratory
outstation, ID2 (ESRF, Grenoble) and at 9.6 (SRS, Daresbury). JM is
funded by Siriraj Hospital Mahidol University Thailand, EYJ is funded
by the Royal Society and the Cancer Research Campaign, GRS and DIS are
funded by the MRC.
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