Current Research Summaries

  • [Article] General and specific requirements of the selective interactions between CSL and its coregulator proteins

    Notch signaling pathway plays a fundamental role in the regulation of various cellular and developmental processes. Ligand binding releases the intracellular domain of Notch receptor (NICD), which interacts with transcription factor CSL to activate transcription of target genes. In the absence of NICD binding, CSL represses gene expression via the direct recruitment of corepressor proteins including SMRT and KyoT2. Structural and functional analyses revealed the molecular basis of these interactions, in which NICD and corepressor proteins competitively bind to β-trefoil domain (BTD) of CSL using a conserved ΦWΦP motif (Φ: any hydrophobic residues). We previously reported that a ΦWΦP motif (1834IWRP1837) of SMRT directly interacts with CSL-BTD. Here, we performed systematic analyses of the binding surface of CSL to SMRT, KyoT2, and NICD using diverse CSL-BTD mutants defective in SMRT binding. Among CSL mutants, the F235, A258 and P260 residues are generally required for these interactions by providing a non-polar binding surface for ΦWΦP motifs. In contrast, L225F, E231G, and E233G mutants evidenced different binding profiles depending on BTD-binders, suggesting these residues as the specific requirements for their interactions. These results demonstrate that residues within and/or outside the core ΦWΦP motifs of coregulators significantly affect high-affinity binding to CSL and that binding interfaces between CSL-BTD and its binders are rather flexible.

  • [Article] Crystal structure of the complex of DUSP15 and its inhibitor reveals novel inhibitor binding interactions

    Protein tyrosine phosphatases (PTPs) dephosphorylate their target proteins in various cell signaling processes and their dephosphorylation activities are associated with cancers, neurological disorders, and diabetes. Although PTPs are considered as promising drug targets, no PTP-targeting drugs are currently on the market. To understand inhibitor binding to PTP proteins, we determined crystal structure of the complex between DUSP15, a member of PTP family and its inhibitor. In the complex structure, extra electron density observed near the active site pocket was fitted to the inhibitor structure. The inhibitor binding site does not overlap the phosphate-binding active site. The novel inhibitor binding interactions involving the P-, D-, and β3-β4 loops indicate an allosteric inhibition mechanism of the inhibitor.

  • [Article] Structural analysis of the non-catalytic zinc binding site in the minor β-carbonic anhydrase CafD

    The fungal zinc metalloenzyme CafD from Aspergillus fumigatus is a minor β-carbonic anhydrase (β-CA) with low CO2 hydration activity. X-ray crystallography revealed that CafD forms a homodimer that possesses a zinc binding site in the dimer interface in addition to the catalytic zinc in the active site. However, the role of this ‘non-catalytic zinc’ has not been elucidated. In this study, we generated single (C39A or E54A) mutants targeting residues that form the non-catalytic zinc coordination sphere of CafD, and analyzed their structural and biochemical characteristics. Contrary to our expectations, these mutations strongly destabilized the three-dimensional fold of CafD, but the native homodimeric assembly was maintained even though the non-catalytic zinc binding site was disrupted. In the C39A mutant, the absence of the noncatalytic zinc permits flipping of a peptide bond, creating extra space for two water molecules that are vital in the formation of a water-mediated hydrogen bond network with the equivalent E54 and R56 residues of the two chains. By contrast, the E54A mutant contains an unexpected disulfide bond formed between the equivalent cysteine residues at position 39, which confers stability to the non-catalytic zinc-deficient CafD. Overall, our results demonstrate that a water-mediated hydrogen bond network and a disulfide bond may compensate for the loss of non-catalytic zinc at the dimer interface of CafD, and thereby contribute to structural integrity and/or thermal stability.

  • [Crystallization] Preliminary crystallographic analysis of the Fab fragment of camrelizumab, a therapeutic antibody against PD-1

    Programmed death 1 (PD-1) is a coinhibitory receptor on the T-cell surface and its primary biological function is to maintain peripheral tolerance by suppressing T-cell activation through the interaction with its ligands PD-L1 and PD-L2. However, cancer cells can evade immunological recognition and destruction by activating coinhibitory pathways through the molecular interactions of PD-1. Recently, the FDA-approved antibodies targeting PD-1 have brought about a significant breakthrough in the treatment of a wide range of cancers, as they can induce durable therapeutic responses. Here, the Fab fragment of camrelizumab, a therapeutic antibody against PD-1, was overexpressed in the periplasmic region of Escherichia coli and purified, and crystallized. The crystal diffracted to 3.25 Å resolution and belonged to the space group C2, with unit cell parameters a = 274.24, b = 77.90, c = 97.16 Å, and β = 101.93˚. An asymmetric unit of the crystal contains four molecules of the Fab fragment of camrelizumab with a VM of 2.51 Å3 Da-1 and a solvent content of 50.96%.

  • [Crystallization] Purification and preliminary analysis of the nuclease and capping domain of the DNA repair exonuclease SbcD from the Grampositive bacteria Staphylococcus aureus

    The SbcCD complex is the bacterial Mre11-Rad50 homolog, and cleaves blocked DNA ends and hairpins by an ATPdependent endo- and exonuclease activities to repair DNA. The SbcC component consists of an ATP-binding cassettetype nucleotide-binding domain and a flanking coiled-coil insertion containing a dimerization motif. The SbcD component consists of a nuclease and capping domain, a linker region, and a helix-loop-helix (HLH) domain. The structural studies have been in Gram-negative bacteria. It remains to be elucidated the action mechanism at the molecular level, especially in Gram-positive bacteria. Here, we studied the nuclease and capping domain of SbcD from the Gram-positive bacteria Staphylococcus aureus. The protein was overexpressed and purified, and its crystals suitable for the structural study were obtained. We collected X-ray diffraction dataset at a resolution of 3.5 Å. The crystals belong to space group P212121, with unit cell parameters a = 72.6, b = 88.9, and c = 115.8 Å. The molecular replacement trials were failed to determine the structure, and thus we are now growing the Se-Met substituted crystals to solve the crystal structure. This structure will give molecular insights into the DNA repair process mediated by the SbcCD complex, in the Gram-positive bacteria.