Crystal Structure of Human TWEAK in Complex with the Fab Fragment of a Neutralizing Antibody Reveals Insights into Receptor Binding.

The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD) to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases

Model of the TWEAK – Fn14 receptor interaction.

<p>A) Side view of the TWEAK trimer showing the solvent accessible electrostatic surface potential (red −4 kT to blue +4 kT). The positively charged patch indicating the possible receptor binding site (dashed ellipse) is covered by the antibody selected for inhibiting TWEAK-Fn14 interaction (cartoon model of Hv in green and Lv in blue). B) Same view as in A with the antibody and TWEAK surface set transparence. After superposition of cytokine-receptor structures APRIL-BCMA (blue; PDB ID 1XU2), APRIL-TACI (brown; PDB ID 1XU1), TALL-BCMA (red; PDB ID 1OQD) and TALL-BAFFR (green; PDB ID 1OQE) the CRD of the receptors co-localize and mark the putative binding site of Fn14 on TWEAK (only the CRD of the receptors is shown as colored cartoon model). C) The NMR model of the Fn14 CRD (blue; PDB ID 2RPJ) is placed at the putative receptor binding site of TWEAK according to the complex structures shown in B. The basic patch is indicated with the dashed ellipse. Only one of the three receptors is shown. D) Stereo view of the modeled TWEAK-Fn14 CRD interface. Upon rigid body and positional refinement of the putative TWEAK-Fn14 CRD complex a dense hydrogen bond network is formed at the interface. The perfect complementarities of charged and hydrophobic patches, as well as the involvement of Fn14 side chains already shown to play an important role in TWEAK binding support this model.</p

Structure of human TWEAK.

<p>A) Ribbon representations of the TWEAK trimer with one protomer colored orange and the symmetry related ones in gray (crystallographic 3-fold axis indicated as black triangle). On the left top view oriented as in 1A with N- and C-Terminus on the top. In the middle side view oriented as in 1B with labeled N- and C-Terminus. In the situation of the uncleaved precursor the membrane is located on top of the molecule. The disulfide bond is highlighted as stick model and beta strands are labeled according to TNF superfamily nomenclature. The dashed lines indicate flexible loops E-F and A-A’’ not visible in the electron density. On the right, bottom view of the TWEAK trimer. B) Solvent accessible electrostatic surface potential (red −4 kT to blue +4 kT) of the TWEAK trimer with the same orientations as in A. Resembling the high pI of TWEAK with 9.62 the complete upper surface is highly positively charged. A second basic patch is located at the side of the TWEAK trimer (dashed ellipse middle picture). This positively charged region is also found in other members of the TNF family (i.e. APRIL, BAFF) and coincides with their receptor binding site. C) Overview of the TWEAK-TWEAK interface as found in the homotrimer in the same orientation and labeled as in A (middle picture). Notable hydrogen bonds involved in the trimerization are indicated as dashed lines with the respective interacting amino acids as sticks. The hydrogen bonds with interacting atoms and distances are listed in the table.</p

Cross blocking assay.

1<p>The Molar Ratio (MR %) was calculated as the quotient of the secondary antibody binding signal to the primary antibody binding signal, both binding to the surface-presented TWEAK ligand.</p

Interaction of the antibody with TWEAK.

<p>A) Ribbon representation of one Fab fragment binding to one TWEAK protomer (orange:TWEAK, blue:light chain, green:heavy chain). B) Stereo representation of the epitope recognition with interacting residues as labeled stick model and important hydrogen bond interaction highlighted as dashed lines. The binding is mainly mediated by CDR loop 1 and 2 of the heavy chain interacting with residues of the loops connecting strands D/E and B’/B and residues of strand G. In addition Y93 of CDR3 of the light chain interacts with a main chain N and stacks with the guanidinium group of R130 of TWEAK. C) Interestingly not only canonical CDR loops are involved in TWEAK binding, but an additional hydrogen bond is formed between light chain R68 of a non CDR loop with D75 of a second subunit of the trimeric TWEAK complex (gray).</p

Biochemical analysis of selected anti TWEAK antibodies.

<p>Biochemical analysis of selected anti TWEAK antibodies.</p

Crystallographic data collection and model refinement statistics.

<p>Crystallographic data collection and model refinement statistics.</p