Nijmegen breakage syndrome 1 (NBS1) forms the MRE11–RAD50–NBS1 complex and is essential for homologous recombination repair of DNA double-strand breaks. NBS1 regulates the localization and activity of the MRN complex. The NBS1 forkhead-associated (FHA) domain represents a potential target for the development of radiosensitizers. However, detailed structural information on the N-terminal region of human NBS1 (hNBS1) is still lacking. Here, the X-ray structure of the hNBS1 FHA–BRCA1 C-terminal (BRCT1)–BRCT2 domains is reported and ligand binding is evaluated using computational docking. A candidate radiosensitizer was predicted to bind at the tip of the FHA domain, indicating a potential interaction site. Substitution of Arg28 with aspartic acid within the FHA domain altered the orientation of neighboring side chains and modified the surface electrostatic properties, supporting its structural and functional importance. These findings provide a structural basis for targeting NBS1 and may facilitate the development of radiosensitizers.
The enzyme phosphoribosyl isomerase A from Mycobacterium tuberculosis (mtPriA) catalyzes two related reactions of bi- and monophosphorylated substrates within the histidine and tryptophan biosynthetic pathways. We have determined the crystal structure of mtPriA with a bound light-controllable 1,2-dithienylethene inhibitor (DTE LC6) at a resolution of 1.95 Å. DTE LC6 contains two terminal phosphate groups, and we found one inhibitor molecule (DTE LC6-1) simultaneously bound to the two known functional phosphate-binding sites of mtPriA. Unexpectedly, another inhibitor molecule (DTE LC6-2) was shown to bind to a hitherto unidentified third phosphate-binding site and thus bridges two crystallographic symmetry-related mtPriA proteins. Our study explains the previously reported inhibitory effect of DTE LC6 on the catalytic activity of mtPriA and raises the question of the significance of the third phosphate-binding site in mtPriA.
The nucleocapsid (N) protein of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) functions in viral RNA binding, genome packaging and ribonucleoprotein (RNP) assembly. Crystal structures were determined of the N-terminal domain (NTD) from crystals grown at pH 8.0 to a resolution of 1.94 Å and of the C-terminal domain (CTD) from crystals grown at pH 5.0 and 8.5 to resolutions of 1.59 and 1.30 Å, respectively. The NTD adopts the canonical coronavirus NTD fold and contains a positively charged cleft corresponding to a previously proposed RNA-binding region. Comparison of the apo NTD structure determined in this study with a previously reported RNA-bound NTD structure revealed local differences in the side-chain conformations of RNA-contacting residues, including Thr49, Arg88, Arg92, Arg107, Tyr109 and Tyr111, whereas the overall domain fold remained essentially unchanged. These observations suggest that RNA binding may be accommodated by local side-chain rearrangements within a pre-existing positively charged cleft rather than by a large-scale conformational change. The CTD forms a domain-swapped dimer mediated by β-hairpins, with a positively charged groove extending across the dimer interface. Superposition of the two CTD structures yielded a root-mean-square deviation (r.m.s.d.) of 0.129 Å, and 11 intersubunit hydrogen bonds were observed at the dimer interface in each structure, indicating that the dimer architecture is conserved under the crystallization conditions examined. Together, these structures provide a framework for further investigation of NTD–RNA recognition, the possible role of the CTD positively charged groove in nucleic acid interactions and N-protein assembly.
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.
