The Catalytic mechanism of decarboxylative hydroxylation of salicylate hydroxylase revealed by crystal structure analysis at 2.5 resolution

Uemura, Takuya*; Kita, Akiko*; Watanabe, Yoshihiko*; Adachi, Motoyasu; Kuroki, Ryota; Morimoto, Yukio*

Biochemical and Biophysical Research Communications, 469(2), p.158 - 163, 2016/01

https://doi.org/10.1016/j.bbrc.2015.11.087

Interaction of double-stranded DNA with polymerized PprA protein from

Adachi, Motoyasu; Hirayama, Hiroshi; Shimizu, Rumi; Sato, Katsuya; Narumi, Issey*; Kuroki, Ryota

Protein Science, 23(10), p.1349 - 1358, 2014/10

https://doi.org/10.1002/pro.2519

Pleiotropic protein promoting DNA repair A (PprA) is a key protein that facilitates the extreme radioresistance of . To clarify the role of PprA in the radioresistance mechanism, the interaction between recombinant PprA expressed in Escherichia coli with several double-stranded DNAs was investigated. In a gel-shift assay, the band shift of supercoiled pUC19 DNA caused by the binding of PprA showed a bimodal distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 M with a Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number of bound PprA molecules estimated by the UV absorption of the PprA-pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was greater than 1.3 M. This result implies that DNA-bound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA.

Biomacromolecular neutron crystallography; Practical methods and utilization of neutron crystallography for understanding protein structure and function

Adachi, Motoyasu; Arai, Shigeki; Hiromoto, Takeshi; Kuroki, Ryota

Hamon, 24(1), p.45 - 49, 2014/02

https://doi.org/10.5611/hamon.24.1_45

Protein structure analysis using neutron diffraction (neutron protein crystallography; NPC) is gaining greater importance in the understanding of structure and function relationships of biological macromolecules such as proteins and DNA. Current developments of neutron diffractometers installed at the JAEA research reactor and pulsed neutron source permit observation of the locations of hydrogen atoms and hydrating water molecules and help understanding of important mechanisms of chemical reactions catalyzed by biological macromolecules. Here, we introduce practical approaches of NPC including sample preparation, crystal growth, structure determination and utilization of information obtained from NPC.

Structure of enzyme-inhibitor complex determined by neutron crystallography

Tamada, Taro; Adachi, Motoyasu; Kurihara, Kazuo; Kuroki, Ryota

Nihon Kessho Gakkai-Shi, 55(1), p.47 - 51, 2013/02

https://doi.org/10.5940/jcrsj.55.47

Neutron crystallography enables us to identify the accurate hydrogen positions in proteins, which play important roles in many chemical reactions in living system. Here we show our results of neutron structure determination of enzymes in complex with its inhibitors which corresponds to transition state analogues. Neutron structure analysis elucidated the detail catalytic reaction of each enzyme by direct observation of hydrogen atoms. Furthermore we would like to introduce a new neutron beam line for neutron structural biology planned at MLF in J-PARC.

Crystallization and preliminary neutron diffraction studies of ADP-ribose pyrophosphatase-I from HB8

Okazaki, Nobuo; Adachi, Motoyasu; Tamada, Taro; Kurihara, Kazuo; Oga, Takushi*; Kamiya, Nobuo*; Kuramitsu, Seiki*; Kuroki, Ryota

Acta Crystallographica Section F, 68(1), p.49 - 52, 2012/01

https://doi.org/10.1107/S1744309111044551

Towards investigation of the inhibitor-recognition mechanisms of drug-target proteins by neutron crystallography

Kuroki, Ryota; Okazaki, Nobuo; Adachi, Motoyasu; Ohara, Takashi; Kurihara, Kazuo; Tamada, Taro

Acta Crystallographica Section D, 66(11), p.1126 - 1130, 2010/11

https://doi.org/10.1107/S0907444910034967

It is generally known that enzymes represent important drug-target proteins. Elucidation of the catalytic function and the molecular-recognition mechanisms of enzymes provides important information for structure-based drug design. Neutron crystallography provides accurate information on the locations of H atoms that are essential in enzymatic function and molecular recognition. Recent examples are described of the structure determination of the drug-target proteins human immunodeficiency virus protease and porcine pancreatic elastase in complex with transition-state analogue inhibitors using the neutron diffractometers for biological crystallography (BIX-3 and BIX-4) installed at the JRR-3 research reactor.

Collaborative use of neutron and X-ray for determination of drug target proteins

Kuroki, Ryota; Tamada, Taro; Kurihara, Kazuo; Ohara, Takashi; Adachi, Motoyasu

Yakugaku Zasshi, 130(5), p.657 - 664, 2010/05

https://doi.org/10.1248/yakushi.130.657

Crystallography enables us to obtain accurate atomic positions within proteins. High resolution X-ray crystallography provides information for most of the atoms comprising a protein, with the exception of hydrogens. Neutron diffraction data can provide information of the location of hydrogen atoms to the structural information determined by X-ray crystallography. Here, we show the recent result of the structural determination of drug-target proteins, porcine pancreatic elastase and human immuno-deficiency virus type-1 protease by both X-ray and neutron diffraction. The structure of porcine pancreatic elastase with its potent inhibitor was determined at room temperature to 1.2 resolution by X-ray diffraction and 1.65 resolution by neutron diffraction. The structure of HIV-PR with its potent inhibitor was also determined to 1.4 resolution by X-ray diffraction and 1.9 resolution by neutron diffraction. Ultra-high resolution structures of both proteins (0.94 and 0.93 , respectively) were also determined by X-ray diffraction at 100 K. The ionization state and the location of hydrogen atoms of the catalytic residue in these enzymes were determined by neutron diffraction. Furthermore, collaborative use of both X-ray and neutron to identify the location of ambiguous hydrogen atoms will be shown.

Structure determination of drug target proteins by neutron crystallography

Tamada, Taro; Adachi, Motoyasu

Radioisotopes, 59(4), p.299 - 308, 2010/04

https://doi.org/10.3769/radioisotopes.59.299

Neutron diffraction data can provide information of the location of hydrogen atoms to the structural information determined by X-ray crystallography. Here, we show the recent results of the structural determination of drug-target proteins, porcine pancreatic elastase and human immuno-deficiency virus type-1 protease by both X-ray and neutron diffraction. The structure of porcine pancreatic elastase with its potent inhibitor was determined to 0.094 nm resolution by X-ray diffraction and 0.165 nm resolution by neutron diffraction. The structure of HIV-PR with its potent inhibitor was also determined to 0.093 nm resolution by X-ray diffraction and 0.19nm resolution by neutron diffraction. The ionization state and the location of hydrogen atoms of the catalytic residue in these enzymes were determined by neutron diffraction.

Structure of HIV-1 protease determined by neutron crystallography

Adachi, Motoyasu; Kuroki, Ryota

Hamon, 19(4), p.214 - 217, 2009/10

To develop HIV-1 protease inhibitors through structure-based drug design, it is necessary to understand the catalytic mechanism and inhibitor recognition of HIV-1 protease. We have determined the crystal structure of HIV-1 protease in complex with KNI-272 to 1.9 resolution by neutron crystallography in combination with 1.4 resolution X-ray diffraction data. The results show that the carbonyl group of hydroxymethylcarbonyl (HMC) in KNI-272 forms a hydrogen bonding interaction with protonated Asp 25 and the hydrogen atom from the hydroxyl group of HMC forms a hydrogen bonding interaction with the deprotonated Asp125. This is the first neutron report for HIV-1/inhibitor complex and shows directly the locations of key hydrogen atoms in catalysis and in the binding of a transition-state analog.

Combined high-resolution neutron and X-ray analysis of inhibited elastase confirms the active-site oxyanion hole but rules against a low-barrier hydrogen bond

Tamada, Taro; Kinoshita, Takayoshi*; Kurihara, Kazuo; Adachi, Motoyasu; Ohara, Takashi; Imai, Keisuke*; Kuroki, Ryota; Tada, Toshiji*

Journal of the American Chemical Society, 131(31), p.11033 - 11040, 2009/07

https://doi.org/10.1021/ja9028846

To help resolve long-standing questions regarding the catalytic activity of the serine proteases the structure of porcine pancreatic elastase has been analyzed by high-resolution neutron and X-ray crystallography. In order to mimic the tetrahedral transition intermediate a peptidic inhibitor was used. A single large crystal was used to collect room-temperature neutron data to 1.65 resolution and X-ray data to 1.20 resolution. Another crystal provided a low-temperature X-ray data set to 0.94 resolution. The neutron data are to higher resolution than previously reported for a serine protease and the X-ray data are comparable with other studies. The neutron and X-ray data show that the hydrogen bond between His57 and Asp102 (chymotrypsin numbering) is 2.60 in length and that the hydrogen-bonding hydrogen is 0.80-0.96 from the histidine nitrogen. This is not consistent with a low-barrier hydrogen which is predicted to have the hydrogen midway between the donor and acceptor atom. The observed interaction between His57 and Asp102 is essentially a short but conventional hydrogen bond, sometimes described as a short ionic hydrogen bond. The neutron analysis also shows that the oxygen of the oxopropyl group of the inhibitor is present as an oxygen anion rather than a hydroxyl group, supporting the role of the "oxyanion hole" in stabilizing the tetrahedral intermediate in catalysis.