Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
-
Communicated by Peter C. Nowell, University of Pennsylvania School of Medicine, Philadelphia, PA, November 27, 2007 (received for review October 4, 2007)
Article Figures & SI
Figures
- Fig. 1.
Overall structure of mGITRL. (a) Ribbon representation of mGITRL monomer structure. The conserved β-strands (A–H) and the extending C-terminus arm with an extra β-strand (I) on the end were labeled. Note that the β-strand F was interrupted. (b) Stereoview of the electron density for the C-terminal arm. The electron density omit map was contoured at 2σ levels.
- Fig. 2.
mGITRL associates as a dimer. (a) Stereoview of mGITRL dimer. Protomers A and B are yellow and pink, respectively. The C-terminus extra β-strand (I) from one protomer joins the β-sheet (A′-A-H-C-F) of the other to form an extended intermolecular β-sheet (I-A′-A-H-C-F). (b) GITRL dimer interface viewed down the 2-fold axis. Residues on the dimeric interface are highlighted in the stick model. Alterative conformations for residues Ile-164 and Met-53 of protomer A and Leu-90, Met-166 and leu169 of protomer B are shown.
- Fig. 3.
Ultracentrifugation analysis of mGITRL. (Upper) Experimental radial concentration profiles (points) and fits (lines) to a monomer–dimer–trimer equilibrium model at three different speeds (15, 25, and 33 K RPM, respectively). (Lower) Calculated weight fractions of monomer (decreasing), dimer (increasing), and trimer (essentially zero) for concentrations covered in the experiment.
- Fig. 4.
Putative 2:2 ligand–receptor complex. (a) Molecular shape of canonical TNF trimer. Green arrow shows the putative receptor binding crevices. (b) mGITRL dimer and putative receptor binding position. (c) Model of the receptor–ligand complex based on the OX40L-OX40 complex. A model for 2:1 would be similar except one of the receptors was removed and hence is not shown.
- Fig. 5.
NF-κB activation with wild-type mGITRL and its C-terminal deletion mutants. Cos cells were transfected with WT, mutant Del1, or mutant Del2 expression plasmids. NF-κB activity was measured by luciferase activity. Generated luciferase activity with each GITRL (WT, D1, or D2)/COS transfectants was compared with that generated by the COS cell transfectant with the empty vector.
- Fig. 6.
Model for mGITRL oligomerization and signaling. mGITRL dimer stabilized by the C-terminus tether shows moderate activity, consistent with our earlier studies (15). Deletion of the anchor β-strand from the C-terminus tethering arm (Del1) is likely to induce conformational readjustments, so that mGITRL might form a trimer similar to OX40L. Thus, the mutant shows higher activity than that of dimer. Complete deletion of the C-terminus arm (Del2) will destabilize both dimers and trimers and therefore lead to a significant reduction in transcription activity.
Data supplements
Zhou et al. 10.1073/pnas.0711206105.
Supporting Information
Files in this Data Supplement:
SI Figure 7
SI Table 1
SI Figure 8
Fig. 7. Structure-based sequence alignment of the mGITRL against other members of the TNFSF. Gray, orange, and green represent residues on the trimeric interface, residues on dimeric interface of GITRL, and residues shared by dimeric and trimeric interface, respectively. Green arrows above the sequences represent conserved b-strands in TNFSF. The extra mini strand (H') unique to mGITRL is shown in pink.
Table 1. Statistics of data collection and refinement
Native
K2PtCl4
HgCl2
Data collection
Space group
P212121
P212121
P212121
Cell dimensions
a, b, c, Å
52.56, 70.40, 71.65
52.65, 69.57, 72.56
52.57, 70.35, 71.70
a, b, g, °
90.00, 90.00, 90.00
90.00, 90.00, 90.00
90.00, 90.00, 90.00
Resolution, Å
1.750(1.810-1.750)
2.300(2.380-2.300)
2.500(2.590-2.500)
Rsym or Rmerge
0.07500(0.3460)
0.09600(0.4970)
0.07900(0.2190)
I / sI
48.56(8.048)
28.64(6.273)
42.63(16.61)
Completeness, %
100.0(100.0)
99.50(99.40)
99.50(99.60)
Redundancy
21.90(21.40)
14.70(14.60)
19.70(20.00)
Refinement
Resolution, Å
50.00-1.750
No. reflections
25,908
Rwork / Rfree
0.1804 / 0.2209
No. atoms
Protein
2,152
Ligand/ion
N/A
Water
252.0
B factors
Protein
17.92
Ligand/ion
N/A
Water
29.97
rmsd
Bond lengths, Å
0.0130
Bond angles, °
1.4050
Values in parentheses are for highest-resolution shell. N/A, not available.
Fig. 8. Superimposition of mGITRL with OX40L. The two subunits of the mGITRL dimer are rendered in yellow and pink, respectively. The OX40L trimer is shown in cyan. When one subunit (yellow) of mGITRL dimer was superimposed onto subunit A of OX40L trimer, the other subunit (pink) of the dimer would take a position between OX40L subunits B and C but slightly toward B.
Structural basis for ligand-mediated mouse GITR activation
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