Kiyohito Kihira

Crystal structure analysis of the translation factor RF3 (release factor 3)

The bacterial translational GTPases release factor RF3 promotes translation termination by recycling RF1 or RF2. Here, we present the crystal structures of RF3 complexed with GDP and guanosine 3′,5′‐(bis)diphosphate (ppGpp) at resolutions of 1.8 and 3.0 Å, respectively. ppGpp is involved in the so‐called “stringent response” of bacteria. ppGpp binds at the same site as GDP, suggesting that GDP and ppGpp are two alternative physiologically relevant ligands of RF3. We also found that ppGpp decelerates the recycling of RF1 by RF3. These lines of evidence suggest that RF3 functions both as a cellular metabolic sensor and as a regulator.

Crystallization and preliminary X-ray analysis of the NAD + -reducing (NiFe) hydrogenase from Hydrogenophilus thermoluteolus TH-1

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Å resolution and belonged to space group C2, with unit-cell parameters a=131.43, b=189.71, c=124.59 Å, β=109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, VM was calculated to be 2.2 Å3 Da(-1), which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.

Crystallization and preliminary X-ray analysis of a class II release factor RF3 from a sulfate-reducing bacterium

Class II release factor 3 (RF3) from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F, which promotes rapid dissociation of a class I release factor, has been overexpressed, purified and crystallized in complex with GDP at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to 1.8 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belongs to space group P1, with unit-cell parameters a = 47.39, b = 82.80, c = 148.29 A, alpha = 104.21, beta = 89.78, gamma = 89.63 degrees . The asymmetric unit contains four molecules of the RF3-GDP complex. The Matthews coefficient was calculated to be 2.3 A(3) Da(-1) and the solvent content was estimated to be 46.6%.

Crystal structure analysis of release factor 3

Crystal structure analysis of release factor 3 Kiyohito Kihira,a Yoshihiro Shimizu,b Yasuhito Shomura,a Masaya Kitamura,c Atsushi Nakagawa,d Tomitake Tsukihara,a Takuya Ueda,e Kozo Ochi,f Yoshiki Higuchi,a aGraduate School of Life Science, Univ. of Hyogo. bQuantitative Biology Center, RIKEN, cGraduate School of Engeneering, Osaka City Univ. dInst. for Protein Research, Osaka Univ. eGraduate School of Frontier Sciences, Univ. of Tokyo. fDepartment of Health Science, Hiroshima Inst. of Tech. (Japan). Email: kihira@protein.osaka-u.ac.jp