Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

Catalytic mechanism and evolutionary characteristics of thioredoxin from Halobacterium salinarum NRC-1.

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from the extremely halophilic archaeon Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content [(Asp + Glu)/(Arg + Lys + His) = 9.0] among known TRXs, was chosen to elucidate the catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main-chain structure of HsTRX-A is similar to those of homologous TRXs; for example, the root-mean-square deviations on Cα atoms were <2.3 Šfor extant archaeal TRXs and <1.5 Šfor resurrected Precambrian TRXs. A unique water network was located near the active-site residues (Cys45 and Cys48) in HsTRX-A, which may enhance the proton transfer required for the reduction of substrates under a high-salt environment. The high density of negative charges on the molecular surface (3.6 × 10-3 e Å-2) should improve the solubility and haloadaptivity. Moreover, circular-dichroism measurements and enzymatic assays using a mutant HsTRX-A with deletion of the long flexible N-terminal region (Ala2-Pro17) revealed that Ala2-Pro17 improves the structural stability and the enzymatic activity of HsTRX-A under high-salt environments (>2 M NaCl). The elongation of the N-terminal region in HsTRX-A accompanies the increased hydrophilicity and acidic residue content but does not affect the structure of the active site. These observations offer insights into molecular evolution for haloadaptation and potential applications in halophilic protein-related biotechnology.

Direct Observation of the Protonation States in the Mutant Green Fluorescent Protein.

Neutron crystallography has been used to elucidate the protonation states for the enhanced green fluorescent protein, which has revolutionized imaging technologies. The structure has a deprotonated hydroxyl group in the fluorescent chromophore. Also, the protonation states of His148 and Thr203, as well as the orientation of a critical water molecule in direct contact with the chromophore, could be determined. The results demonstrate that the deprotonated hydroxyl group in the chromophore and the nitrogen atom ND1 in His148 are charged negatively and positively, respectively, forming an ion pair. The position of the two deuterium atoms in the critical water molecule appears to be displaced slightly toward the acceptor oxygen atoms according to their omit maps. This displacement implies the formation of an intriguing electrostatic potential realized inside of the protein. Our findings provide new insights into future protein design strategies along with developments in quantum chemical calculations.

The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions.

Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1-163 residues of hTPO (hTPO163 ) including the N-terminal active site domain (1-153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332 ). In order to clarify the structural and physicochemical properties of hTPO163 , circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0% for helix and -16.7% for ß-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm ) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154-332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography.

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Šresolution) and X-ray (1.1 Šresolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%-75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.

Polypentagonal ice-like water networks emerge solely in an activity-improved variant of ice-binding protein.

Polypentagonal water networks were recently observed in a protein capable of binding to ice crystals, or ice-binding protein (IBP). To examine such water networks and clarify their role in ice-binding, we determined X-ray crystal structures of a 65-residue defective isoform of a Zoarcidae-derived IBP (wild type, WT) and its five single mutants (A20L, A20G, A20T, A20V, and A20I). Polypentagonal water networks composed of ∼50 semiclathrate waters were observed solely on the strongest A20I mutant, which appeared to include a tetrahedral water cluster exhibiting a perfect position match to the [Formula: see text] first prism plane of a single ice crystal. Inclusion of another symmetrical water cluster in the polypentagonal network showed a perfect complementarity to the waters constructing the [Formula: see text] pyramidal ice plane. The order of ice-binding strength was A20L < A20G < WT < A20T < A20V < A20I, where the top three mutants capable of binding to the first prism and the pyramidal ice planes commonly contained a bifurcated γ-CH3 group. These results suggest that a fine-tuning of the surface of Zoarcidae-derived IBP assisted by a side-chain group regulates the holding property of its polypentagonal water network, the function of which is to freeze the host protein to specific ice planes.

Neutron structure of the T26H mutant of T4 phage lysozyme provides insight into the catalytic activity of the mutant enzyme and how it differs from that of wild type.

T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the ß-1,4-glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09-Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated Nɛ2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water-binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high-resolution X-ray structure of T26H at 1.04-Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (-1.52 ± 0.05 kJ mol(-1)  K(-1) ) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 ∼ 0.25 kJ mol(-1)  K(-1) ) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

Structure of a highly acidic ß-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs(+)-selective binding site.

Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic ß-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Šin space group P31 using X-ray crystallography. Moreover, the locations of bound Sr(2+) and Cs(+) ions were identified by anomalous X-ray diffraction. The location of one Cs(+)-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na(+) (90 mM Na(+)/10 mM Cs(+)). From an activity assay using isothermal titration calorimetry, the bound Sr(2+) and Cs(+) ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs(+)-binding site provides important information that is useful for the design of artificial Cs(+)-binding sites that may be useful in the bioremediation of radioactive isotopes.

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum.

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.