Neutron diffraction study to elucidate reaction mechanism of RNase A

Ribonuclease A (RNase A) is a pyrimidine-specific endoribonuclease that claves and hydrolyzes single-stranded RNA in two distinct steps. The mechanism of the cleavage reaction catalyzed by RNase A involves two key histidine residues, His12 and His119. It is important to know the protonation states of them in order to understand the hydrolysis mechanism of RNase A. Neutron protein crystallography is a powerful technique for solving the problems. In previous reports, the protonation states of them for RNase A complexed with phosphate ion, uridine vanadate and phosphate free one have been investigated by neutron diffraction analysis [1-3]. In this study, neutron diffraction analysis of phosphate free RNase A has been carried out with high resolution and completeness data set in order to clarify the protonation states of two active site histidine residues, and to elucidate the detailed mechanism of the cleavage reaction. Neutron diffraction data of bovine pancreatic RNase A were collected by IBARAKI biological crystal diffractometer iBIX in J-PARC. The structure was determined by joint neutron and X-ray structure refinement. The final values of Rcryst and Rfree were 19.5% and 22.0%, respectively, for completeness of 86.7% to a resolution of 1.4Å. The structure with high reliability and good data statistics could be obtained by comparing with the already-reported one [3]. We calculated |Fo|-|Fc| neutron scattering length density map after omitting Dδ1 and Dε2 of His12 and His119 in order to confirm the protonation states of them. These omit maps indicated that His12 is completely singly protonated and Hi119 is doubly protonated. The protonation states of them are consistent with those in the first step of the putative mechanism of catalysis by RNase A. We could also observed a D atom of water molecule which is hydrogen bonded to Nε2 of His12.

Neutron crystallographic analysis of human α-thrombin using iBIX at J-PARC

α-Thrombin is a serine protease, which plays the central role in the coagulation system. Investigating the protonation states of the enzyme is useful to reveal the reaction mechanism and to design anticoagulant drugs. We have studied the protonation states of the α-Thrombin-bivalirudin complex using neutron diffraction method. The complex is regarded as the enzyme-product complex, because the hydrolyzed bivalirudin fragments keep staying in the binding sites in the crystal. Previously we had performed a neutron crystallographic analysis of α-Thrombin-bivalirudin complex at pD 5.0 (space group P21212) at 2.8 Å resolution.[1] To observe the protonation states of the active site more clearly, we carried out time-of-flight neutron diffraction experiments for a different crystal form of this complex (space group C2 at pD 7.9) using IBARAKI biological crystal diffractometer, iBIX, installed in J-PARC. Using improved 30 neutron detectors with high-efficiency, we have succeeded in collecting the reflections at around 2.0 Å resolution. XN-joint refinements were performed using PHENIX program. The neutron scattering length OMIT map showed a density on the hydroxyl group of serine 195, which could be a deuterium. Since the density was not observed for P21212 crystal at pD 5.0 and the position was too far from an acceptor atom to form a stiff hydrogen bond, currently we are confirming the result. In this presentation, details of the neutron crystallographic analysis and the comparison between the structures, especially, the protonation states of amino acid residues in the active site of the complex at pD 5.0 and pD 7.9 will be given.

Neutron diffractometer covering protein crystals with large unit cell at J-PARC

The structural information of hydrogen atoms and hydration waters obtained by neutron protein crystallography is expected to contribute to elucidation of protein function and its improvement. However, many proteins, especially membrane proteins and protein complexes, have larger molecular weight and then unit cells of their crystals have larger volume, which is out of range of measurable unit cell volume for conventional diffractometers. Therefore, our group had designed the diffractometer which can cover such crystals with large unit cell volume (target lattice length: 250 Å). This diffractometer is dedicated for protein single crystals and has been proposed to be installed at J-PARC (Japan Proton Accelerator Research Complex). Larger unit cell volume causes a problem to separate spots closer to each other in spatial as well as time dimension in diffraction images. Therefore, our proposed diffractometer adopts longer camera distance (L2 = 800mm) and selects decoupled hydrogen moderator as neutron source which has shorter pulse width. Under the conditions that L1 is 33.5m, beam divergence 0.4⁰ and crystal edge size 2mm, this diffractometer is estimated to be able to resolves spots diffracted from crystals with a lattice length of 220 Å in each axis at d-space of 2.0 Å. In order to cover large neutron detecting area due to long camera distance, novel large-area detector (larger than 300mm × 300mm) with a spatial resolution of better than 2.5mm is under development. More than 40 these detectors plan to be installed, providing the total solid angle coverage of larger than 33%. For neutron guide, ellipsoidal supermirror is considered to be adopted to increase neutron flux at the sample position. The final gain factor of this diffractometer is estimated to be about 20 or larger as compared with BIX-3/4 diffractometers operated in the research reactor JRR-3 at JAEA (Japan Atomic Energy Agency) [1,2].