Shigetoshi Sugio

Three-dimensional structure of the ribonuclease t1 · 3'-guanylic acid complex at 2.6 Å resolution

X‐ray crystallography Ribonuclease T1 3‐Guanylic acid Ribonuclease T1 · 3guanylic acid complex Specific recogniton Disordered ribose

pH-induced change in nucleotide binding geometry in the ribonuclease T1-2'-guanylic acid complex: Refinement of X-ray structure at 1.9 Å resolution

At pH 4.0, the RNase T1‐2GMP complex (1) crystallizes isomorphously with the isoenzyme complex (2) (Heinemann, U. and Saenger, W., 1982, Nature 299, 27‐31). The X‐ray structure of 1 was refined with 1.9 Å data to R = 0.195. Polypeptide folding is similar in 1 and 2. However, the sugar pucker of 2‐GMP is 2‐endo (3endo in 2), and guanine binding involves four hydrogen bonds in 1, which all differ from the two bonds in 2. Phosphate contacts Glu58, Arg77, Tyr38 in 1, but His40 in 2. These changes are not due to differences in sequence between the mother‐ and isoenzyme (Gln25‐Lys) but are associated with pH changes leadingto an inactive enzyme structure.

Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein

Tau-tubulin kinase 2 (TTBK2) is a Ser/Thr kinase that putatively phosphorylates residues Ser208 and Ser210 (numbered according to a 441-residue human tau isoform) in tau protein. Functional analyses revealed that a recombinant kinase domain (residues 1-331) of human TTBK2 expressed in insect cells with a baculovirus overexpression system retains kinase activity for tau protein. The kinase domain of TTBK2 was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 55.6, b = 113.7, c = 117.3 A, alpha = beta = gamma = 90.0 degrees. Diffraction data were collected to 2.9 A resolution using synchrotron radiation at BL24XU of SPring-8.

Crystal structure of human dual specificity phosphatase, JNK stimulatory phosphatase‐1, at 1.5 Å resolution

Human JNK stimulatory phosphatase‐1 (JSP‐1) is a novel member of dual specificity phosphatases. A C‐terminus truncated JSP‐1 was expressed in Escherichia coli and was crystallized using the sitting‐drop vapor diffusion method. Thin‐plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit‐cell parameters a = 84.0 Å, b = 49.3 Å, c = 47.3 Å, and β = 119.5°, and diffract up to 1.5 Å resolution at 100 K. The structure of JSP‐1 has a single compact (α/β) domain, which consists of six α‐helices and five β‐strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP‐loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP‐1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP‐1 has no loop corresponding to the Lys120‐loop of PTP1B, and tryptophan residue corresponding to the substrate‐stacking in PTP1B is substituted by alanine residue in JSP‐1. Proteins 2007.