EIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O₂ binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His-183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6, H7, and loop L7) of the globin domain, and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI⁻ cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.

Caspase‐2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase‐2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14‐3‐3, which recognizes two phosphoserines located in the linker between the CARD and the p19 domains of the caspase‐2 zymogen. However, the structural details of this interaction and the exact role of 14‐3‐3 in the regulation of caspase‐2 activation remain unclear. Moreover, the caspase‐2 region with both 14‐3‐3 binding motifs also contains the nuclear localization sequence, thus suggesting that 14‐3‐3 binding may regulate the subcellular localization of caspase‐2. Here, we report a structural analysis of the 14‐3‐3ζ:caspase‐2 complex using a combined approach based on small angle X‐ray scattering, NMR, chemical crosslinking and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase‐2 and 14‐3‐3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase‐2 are positioned within the central channel of the 14‐3‐3 dimer and stabilized through interactions with the C‐terminal helices of both 14‐3‐3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase‐2 activation by dimerization, is sterically occluded by the 14‐3‐3 dimer, thereby likely preventing caspase‐2 activation. In addition, 14‐3‐3 protein binding to caspase‐2 masks its nuclear localization sequence. Therefore, our results suggest that 14‐3‐3 protein binding to caspase‐2 may play a key role in regulating caspase‐2 activation.