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.

Radical doping and high-pressure freezing in an advanced neutron crystallography

The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt) as an advanced NPC. Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of polarized proton) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this poster, two developing fundamental studies to realize ppt will be presented; 1) radical doping into protein crystals that facilitates sample electron polarization, which was analyzed by X-ray crystallography, liquid-chromatography/mass-spectrometry (LC/MS) and electron spin resonance (ESR) measurement, 2) high-pressure flash freezing performed especially using a new machine of HPC-201 (ADC Inc.), which has the advantage of making bulk water amorphous without destroying the single large crystal, may easily realize the low temperature environment of crystal at around 1K. The former results were that radical molecules distributed non-specifically around proteins, and that they were included in protein crystal to some extent [1]. These are a favorable tendency for better proton polarization.

Neutron Structure of Phycocyanobilin:oxidoreductase Complexed with Biliverdin

Phytobilins are linear tetrapyrrole compounds used as chromophore for light harvesting and photoreceptor proteins in higher plants, algae, and cyanobacteria. Phytobilins are synthesized from biliverdin IX(alpha) (BV). Phycocianobilin:oxidoreductase (PcyA) is an enzyme to produce phycocyanobilin (PCB) used as chromophore for light harvesting and photoreceptor proteins. PcyA is unique because it catalyzes the reduction of BV by two sequential steps; the first step is the reduction of the vinyl of the BV D-ring to produce 18(1)-18(2)-dihydrobiliverdin (18EtBV), and the second step is the reduction of the A-ring. In these reduction steps, four hydrogen atoms are delivered to BV. The earlier studies showed that the carboxyl group of Asp105 showed dual conformations. This has been attributed to the difference of its protonation states. The catalytically essential His88 was suggested to be protonated (i. e. His88 is a proton donor) to donate the proton to BV. BVH+ (N-protonated) state, in which four pyrrole N atoms of BV were fully protonated, was proposed to be partially formed when BV was bound to PcyA. Further, another tautomeric BVH+ state in which three of four pyrrole N atoms of BV were protonated and the lactam (C=O) group of BV D-ring was protonated as lactim (C-OH; O-protonated) was proposed. Additionally, newly identified water molecule near BV has been suggested to be a proton donor. To elucidate the H atom positions of these molecules, we determined the neutron crystal structure of the PcyA-BV complex at 1.95 Å resolution. Crystal with approximately 2.2 X 1.8 X 0.8 mm3 size, which was soaked into the deuterium-exchanged crystallization solution, was used in the diffraction experiment. The neutron diffraction intensity data was collected using IBARAKI Biological Crystal Diffractometer (iBIX) in J-PARC. In this conference, we report the protonation states of catalytically important residues and BV as well as orientations of water molecules in the PcyA-BV complex.

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.