Peptidyl-tRNA hydrolase (Pth2) hydrolyzes peptidyl-tRNA, an immature product of aborted translation, into peptide and tRNA, thereby maintaining cellular protein synthesis through peptide release and tRNA recycling. Here, we present the crystal structure of Pth2 from Candidatus Lokiarchaeum sp. GC14_75 (LokiPth2) at 2.12 Å resolution. This is the first structure of Pth2 from a lineage within Promethearchaeati, a kingdom of archaea closely related to eukaryotes. The structure reveals that LokiPth2 forms a homodimer and closely resembles Pth2 structures from other species. However, LokiPth2 exhibits two prominent structural differences: a short helix around the catalytic center, which is absent in other Pth2s, and a distinct orientation of the C-terminal helix. Detailed comparative structural analysis suggests that these regions may regulate enzymatic activity and substrate binding, respectively. Furthermore, the corresponding regions in other Pth2s also exhibit high flexibility, suggesting that similar mechanisms may be conserved among Pth2s. To gain insights into the growth environment of Candidatus Lokiarchaeum sp. GC14_75, we assess the optimal temperature for the catalytic reaction of LokiPth2, which suggests that Candidatus Lokiarchaeum sp. GC14_75 inhabits moderately thermophilic environments.
The Anticalin CL31d, an engineered lipocalin protein previously designed to specifically bind rare-earth and related metal ions as chelate complexes with p-NH2-Bn-CHX-A″-DTPA (DTPA-NH2), was subjected to structural and binding studies with a series of 11 different MIII·DTPA-NH2 complexes. These complexes include various lanthanide and main-group metal(III) ions whose radioisotopes are useful in nuclear medicine, with ionic radii ranging from 0.62 to 1.03 Å. Binding activities of the Anticalin for the MIII·DTPA-NH2 complexes were quantified by fluorescence titration (probing the intrinsic Tyr/Trp emission), revealing Kd values of 0.8-2.4 nM for most of the lanthanide ions investigated (rion = 0.75-0.96 Å), but showing markedly reduced affinities towards the small and large main-group metal ions Ga3+ (Kd = 15.3 nM) and Bi3+ (Kd = 47.8 nM), respectively. The crystal structures of six representative MIII·DTPA-NH2 complexes bound to the Anticalin were solved at high resolution (1.5-1.8 Å) using synchrotron X-ray diffraction. Superposition onto the previously described Anticalin CL31 with bound Y3+·DTPA-SCN indicated an essentially invariant conformation both for the binding protein and its metal-chelate ligands, including conserved hydrogen bonds, and a surprisingly uniform ninefold metal coordination via five carboxylate groups, three N atoms and one water molecule. However, there were two exceptions: the small Sc3+ ion appeared to be coordinated only eightfold with the DTPA-NH2 chelator but lacking the water ligand, whereas no metal electron density was observed for the Ga3+ ion, in line with its known noncanonical DTPA complex geometry. Interestingly, in this case a water molecule was detected at the expected position of the central metal ion within the protein-bound DTPA-NH2 chelator. Our investigation of the influence of chelate geometry on complex stability establishes the Anticalin CL31d as a small and robust universal binding protein for medically relevant MIII·DTPA complexes with surprisingly broad tolerance towards varying ionic radii, thus enabling flexible radionuclide-targeting strategies in nuclear medicine.
The Gly/N-degron pathway is a branch of the proteasomal degradation pathway that specifically targets proteins initiated with an N-terminal glycine. The E3 ligase substrate adaptors ZYG11B and ZER1 have been identified as being responsible for recognizing the target proteins of the Gly/N-degron pathway. Previously, it has been shown that the Gly/N-degron pathway activates the human NLRP1 inflammasome by degrading the autoinhibitory N-terminal fragment of NLRP1 after cleavage by the enteroviral 3C protease. However, the recognition of the NLRP1 Gly/N-degron is not yet fully understood. Here, we determined the X-ray crystal structure of ZER1 bound to the NLRP1 Gly/N-degron at a resolution of 2.2 Å. The structural information revealed that ZER1 uses its ARM repeats to form a conserved cavity that engages the N-terminal glycine (G1) through hydrogen bonds to Asp556, Asn597 and Glu600. Structural comparisons show a shared recognition mode for Gly/N-degrons despite subtle differences in side-chain interactions. However, ZER1 exhibits weaker affinity for the NLRP1 Gly/N-degron than ZYG11B, likely due to distinct local environments surrounding position 3. This study elucidates the molecular basis of NLRP1 recognition by ZER1 and provides insights into targeting this pathway in inflammatory diseases.
NKX2-1 (thyroid transcription factor 1, TTF-1) is a homeodomain transcription factor that plays critical roles in the development and function of the thyroid, lung and forebrain. Here, we report the crystal structure of the NKX2-1 homeodomain bound to a 19 bp DNA duplex containing two palindromically arranged NK2-recognition motifs, refined to 3.26 Å resolution. The structure reveals two homeodomains bound to a single DNA duplex and demonstrates that the overall fold and DNA-binding interactions are highly conserved relative to those of NKX2-5. Comparison with NKX2-5 further shows that the amino-acid residues that differ between the two homeodomains are located away from the protein-DNA interface, suggesting that functional differences between these transcription factors are unlikely to arise from distinct DNA-recognition mechanisms. These findings provide a structural framework for understanding DNA recognition by NKX2-1 and for interpreting the effects of pathogenic variants within its homeodomain.
L-Galactose dehydrogenase plays a key role in the biosynthesis of L-ascorbic acid (vitamin C) in plants. Here, we report crystal structures of rice (Oryza sativa) L-galactose dehydrogenase in both apo and NAD+-bound forms at 1.2 and 1.8 Å resolution, respectively. Rice L-galactose dehydrogenase adopts a conserved (β/α)8-barrel fold with a well preserved NAD+-binding cleft, as in other plant L-galactose dehydrogenases. Structural comparisons revealed conformational flexibility in the first and second loop regions, which form the lid and side wall of the NAD+-binding site. These loops undergo significant rearrangement upon NAD+ binding, likely facilitating cofactor and substrate uptake. These findings suggest a flexible loop-mediated regulation of cofactor and substrate access.
