Sachiko Toma

Crystal structure of the IL-15-IL-15Ralpha complex, a cytokine-receptor unit presented in trans

Interleukin 15 (IL-15) and IL-2, which promote the survival of memory CD8+ T cells and regulatory T cells, respectively, bind receptor complexes that share β- and γ-signaling subunits. Receptor specificity is provided by unique, nonsignaling α-subunits. Whereas IL-2 receptor-α (IL-2Rα) is expressed together in cis with the β- and γ-subunits on T cells and B cells, IL-15Rα is expressed in trans on antigen-presenting cells. Here we present a 1.85-Å crystal structure of the human IL-15–IL-15Rα complex. The structure provides insight into the molecular basis of the specificity of cytokine recognition and emphasizes the importance of water in generating this very high-affinity complex. Despite very low IL-15–IL-2 sequence homology and distinct receptor architecture, the topologies of the IL-15–IL-15Rα and IL-2–IL-2Rα complexes are very similar. Our data raise the possibility that IL-2, like IL-15, might be capable of being presented in trans in the context of its unique receptor α-chain.

The crystal structures of semi-synthetic aequorins

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. Semi‐synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca2+. To understand the structural basis of the Ca2+‐sensitivity, we determined the crystal structures of four semi‐synthetic aequorins (cp‐, i‐, br‐ and n‐aequorins) at resolutions of 1.6–1.8 Å. In general, the protein structures of these semi‐synthetic aequorins are almost identical to native aequorin. Of the four EF‐hand domains in the molecule, EF‐hand II does not bind Ca2+, and the loop of EF‐hand IV is clearly deformed. It is most likely that the binding of Ca2+ with EF‐hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i‐, br‐, and n‐aequorins have bulky 2‐substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca2+ to aequorin. In cp‐aequorin, the cyclopentylmethyl group that substitutes for the original 8‐benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light‐emitting reaction. The differences of various semi‐synthetic aequorins in Ca2+‐sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca2+‐binding.

Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP.

Human MutT homologue 1 (hMTH1) hydrolyzes a variety of oxidized purine nucleoside triphosphates, including 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP and 8-oxo-dATP, to their corresponding nucleoside monophosphates, while Escherichia coli MutT possesses prominent substrate specificity for 8-oxoguanine nucleotides. Three types of crystals were obtained corresponding to the following complexes: selenomethionine-labelled hMTH1 with 8-oxo-dGMP (SeMet hMTH1-8-oxo-dGMP), hMTH1 with 8-oxo-dGMP (hMTH1-8-oxo-dGMP) and hMTH1 with 2-oxo-dATP (hMTH1-2-oxo-dATP). Crystals of the SeMet hMTH1-8-oxo-dGMP complex belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 45.8, c = 153.6 A, and diffracted to 2.90 A. Crystals of hMTH1-8-oxo-dGMP and hMTH1-2-oxo-dATP belong to space groups P2(1) and P2(1)2(1)2(1), with unit-cell parameters a = 34.0, b = 59.0, c = 65.9 A, beta = 90.7 degrees and a = 59.2, b = 67.3, c = 80.0 A, respectively. Their diffraction data were collected at resolutions of 1.95 and 2.22 A, respectively.

Crystallization and preliminary X-ray analysis of human MTH1 with a homogeneous N-terminus

Human MTH1 (hMTH1) is an enzyme that hydrolyses several oxidized purine nucleoside triphosphates to their corresponding nucleoside monophosphates. Crystallographic studies have shown that the accurate mode of interaction between 8-oxoguanine and hMTH1 cannot be understood without determining the positions of the H atoms, as can be observed in neutron and/or ultrahigh-resolution X-ray diffraction studies. The hMTH1 protein prepared in the original expression system from Escherichia coli did not appear to be suitable for obtaining high-quality crystals because the hMTH1 protein had heterogeneous N-termini of Met1 and Gly2 that resulted from N-terminal Met excision by methionine aminopeptidase from the E. coli host. To obtain homogeneous hMTH1, the Gly at the second position was replaced by Lys. As a result, mutant hMTH1 protein [hMTH1(G2K)] with a homogeneous N-terminus could be prepared and high-quality crystals which diffracted to near 1.1 Å resolution using synchrotron radiation were produced. The new crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.36, b = 47.58, c = 123.89 Å.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA.

The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Downs syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 A, beta = 101.7 degrees and a V(M) value of 3.56 A(3) Da(-1). Diffraction data were collected to a resolution of 3.0 A.