Natural Conformational Sampling of Human TNFα Visualized by Double Electron-Electron Resonance

Abstract

Double electron-electron resonance in conjunction with site-directed spin labeling has been used to probe natural conformational sampling of the human tumor necrosis factor α trimer. We suggest a previously unreported, predeoligomerization conformation of the trimer that has been shown to be sampled at low frequency. A model of this trimeric state has been constructed based on crystal structures using the double-electron-electron-resonance distances. The model shows one of the protomers to be rotated and tilted outward at the tip end, leading to a breaking of the trimerous symmetry and distortion at a receptor-binding interface. The new structure offers opportunities to modulate the biological activity of tumor necrosis factor α through stabilization of the distorted trimer with small molecules.

Figure 1

Structure of MTSSL attached to a cysteine side group.

Figure 2

Positions of the two residues I83 (a) and T77 (b) on TNFα that were substituted with cysteines for labeling with MTSSL (PDB: 1TNF). In (a), I83 is shown in yellow, and in (b), T77 is shown in pink.

Figure 3

Crystal structure of MTSSL-labeled T77C apo-TNFα at 1.4 Å resolution (PDB: 5UUI).

Figure 4

Q-band DEER measurements of T77C (a and b) and I83C (d and e) human TNFα. Form factor (blue) and fit (black) are in (a) and (d), and the distance distribution is in (b) and (e). Rotamers calculated in MMM from PDB: 5UUI are shown for T77C and I83C in (c) and (f), respectively, and P(r) values calculated in MMM are shown by red solid peaks in (b) and (e). The inset in (b) is the −2σ standard deviation pooled from nine data sets (35). Peaks labeled i–iv are discussed in the main text. Acquisition conditions are in Materials and Methods.

Figure 5

New model of TNFα showing a predeoligomerization conformation revealed by DEER, with protomer A in cyan, protomer B in green, and protomer C in red viewed from the tip region. T77 residues are highlighted in pink and I83 residues are highlighted in yellow. The 4.5 Å increase in distance between T77 positions at one interface is shown by the arrows in (b) and (f). In (c)–(f), dotted lines indicate apo distances and the solid line indicates the DEER-modeled distance, which differs from apo.