Kuehnemuth, R. | Search bioRxiv

bioRxiv2026

CD95/Fas Apoptosis Signal Initiation Depends on the Ligand Oligomerization State and Formation of Small Ligand-Receptor Complexes

The death receptor cluster of differentiation 95 (CD95 / Fas) is an important inducer of apoptotic activity in tumor cells, but may exhibit differential cell responses (ranging from predominantly apoptosis up to proliferation) when stimulated by its CD95 ligand (CD95L/FasL). How ligand form and receptor-ligand stoichiometry shape these divergent outcomes remain unresolved. To define structural and functional determinants of CD95 signaling, we systematically compared the oligomerization and activity of native CD95L with CD95L variants, where ligand trimers were stabilized either by a FLAG-tag for subsequent monoclonal antibody (mAb) crosslinking or by genetically fused isoleucine zippers (IZ). Apoptotic activity varied markedly with both ligand concentration and ligand variant type. To quantify receptor-ligand stoichiometry at the cell membrane, we combined quantitative Stimulated Emission Depletion (qSTED) nanoscopy, simulations, and biochemical assays. Across conditions, signaling complexes consisted of small oligomeric assemblies, with up to three CD95 receptors engaging a trimeric ligand. Despite similar stoichiometry, biochemical measurements revealed substantial differences in ligand oligomerization and binding affinity, with IZ-CD95L exhibiting markedly higher affinity than FLAG-CD95L (KD of 0.81 nM and 18.4 nM, respectively). Dimeric/trimeric CD95 formed by CD95L complexation or bridged CD95 by mAb enabled flexible intracellular FADD linkage for enhanced signaling.These results indicate that apoptotic potency is governed by the proximity of CD95 receptors, achieved through receptor bridging as well as by a stabilized trimeric ligand, both of which enhance receptor recruitment and binding avidity. Our findings provide mechanistic insight into CD95 signaling and suggest strategies for optimized apoptosis-inducing therapeutics. Significance StatementThe CD95/Fas receptor-ligand system is a central regulator of programmed cell death, yet the earliest molecular events of CD95 activation remain unclear. We systematically compared different CD95L/FasL variants and show that ligand architecture critically shapes receptor engagement and death signaling. An engineered FasL with an isoleucine zipper exhibited larger trimeric lig- and fractions, stability, and receptor binding, resulting in stronger apoptotic activity. Super-resolution imaging and simulations indicate that CD95 is mainly monomeric at the membrane and forms small oligomers after ligand binding. The flexibility of intracellular FADD linkage trigerred by dimeric/trimeric or bridged CD95 enables more intense signaling. These findings clarify how ligand structure governs CD95 activation and provide design principles for death receptor-targeted therapeutics.

biophysics↗

bioRxiv2024

Multi-state kinetics of the syringe-like injection mechanism of Tc toxins

Tc toxins are virulence factors of many insects and human pathogenic bacteria. They attach as soluble prepores to receptors on host cells and following acidification in the late endosome, perforate the cell membrane like a syringe to translocate toxic enzymes into the host cell through their pore-forming channel. Although this complex transformation has been structurally well studied, the functional aspects of this large-scale rearrangement, such as the reaction pathway with possible intermediate states and the resulting temporal evolution have remained elusive. Here, we used an integrated biophysical approach to monitor the prepore-to-pore transition and found that it takes [~]28 h when induced by high pH in the absence of other factors. In the presence of liposomes, an increasingly high pH or receptors, such as heparin or Vsg, the probability to transform prepores to pores increases by a factor of up to 4. This effect can also be mimicked by biotinylation or site-directed mutagenesis of the shell, demonstrating that shell destabilization is a crucial step in prepore-to-pore transition. We show that shell opening is a heterogeneous process with transition times ranging from 60 ms to 1.6 s and resolve three sequential intermediate states: an initial transient intermediate during shell destabilization, a first stable intermediate where the receptor-binding domains on the shell rearrange and a second stable intermediate with an open shell. In contrast, the ejection of the pore-forming channel from the open shell is highly cooperative with a transition time of < 60 ms. This detailed knowledge of the Tc toxin mechanism of action, even in the absence of receptors, is important for the future application of Tc toxins as biomedical devices or biopesticides.

biochemistry↗