Crystal structure and DNA-binding property of the ATPase domain of bacterial mismatch repair endonuclease MutL from Aquifex aeolicus.

DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-ß sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule.

Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

Crystal structure and substrate-binding mode of GH63 mannosylglycerate hydrolase from Thermus thermophilus HB8.

Glycoside hydrolase family 63 (GH63) proteins are found in eukaryotes such as processing α-glucosidase I and also many bacteria and archaea. Recent studies have identified two bacterial and one plant GH63 mannosylglycerate hydrolases that act on both glucosylglycerate and mannosylglycerate, which are compatible solutes found in many thermophilic prokaryotes and some plants. Here we report the 1.67-Å crystal structure of one of these GH63 mannosylglycerate hydrolases, Tt8MGH from Thermus thermophilus HB8, which is 99% homologous to mannosylglycerate hydrolase from T. thermophilus HB27. Tt8MGH consists of a single (α/α)6-barrel catalytic domain with two additional helices and two long loops which form a homotrimer. The structures of this protein in complexes with glucose or glycerate were also determined at 1.77- or 2.10-Å resolution, respectively. A comparison of these structures revealed that the conformations of three flexible loops were largely different from each other. The conformational changes may be induced by ligand binding and serve to form finger-like structures for holding substrates. These findings represent the first-ever proposed substrate recognition mechanism for GH63 mannosylglycerate hydrolase.

Evidence for ATP-dependent structural rearrangement of nuclease catalytic site in DNA mismatch repair endonuclease MutL.

DNA mismatch repair (MMR) greatly contributes to genome integrity via the correction of mismatched bases that are mainly generated by replication errors. Postreplicative MMR excises a relatively long tract of error-containing single-stranded DNA. MutL is a widely conserved nicking endonuclease that directs the excision reaction to the error-containing strand of the duplex by specifically nicking the daughter strand. Because MutL apparently exhibits nonspecific nicking endonuclease activity in vitro, the regulatory mechanism of MutL has been argued. Recent studies suggest ATP-dependent conformational and functional changes of MutL, indicating that the regulatory mechanism involves the ATP binding and hydrolysis cycle. In this study, we investigated the effect of ATP binding on the structure of MutL. First, a cross-linking experiment confirmed that the N-terminal ATPase domain physically interacts with the C-terminal endonuclease domain. Next, hydrogen/deuterium exchange mass spectrometry clarified that the binding of ATP to the N-terminal domain induces local structural changes at the catalytic sites of MutL C-terminal domain. Finally, on the basis of the results of the hydrogen/deuterium exchange experiment, we successfully identified novel regions essential for the endonuclease activity of MutL. The results clearly show that ATP modulates the nicking endonuclease activity of MutL via structural rearrangements of the catalytic site. In addition, several Lynch syndrome-related mutations in human MutL homolog are located in the position corresponding to the newly identified catalytic region. Our data contribute toward understanding the relationship between mutations in MutL homolog and human disease.

Crystal structure of the tandem-type universal stress protein TTHA0350 from Thermus thermophilus HB8.

The genome sequence of an extremely thermophilic bacterium, Thermus thermophilus HB8, revealed that TTHA0350 is a tandem-type universal stress protein (Usp) consisting of two Usp domains. Usp proteins, which are characterized by a conserved domain consisting of 130-160 amino acids, are inducibly expressed under a large number of stress conditions. The N-terminal domain of TTHA0350 contains a motif similar to the consensus ATP-binding one (G-2 x-G-9x-G-(S/T)), but the C-terminal one seems to lack the consensus motif. In order to determine its structural properties, we determined the crystal structures of TTHA0350 in the unliganded form and TTHA0350•2ATP at 2.50 and 1.70 Šresolution, respectively. This is the first structure determination of a Usp family protein in both unliganded and ATP-liganded forms. TTHA0350 is folded into a fan-shaped structure which is similar to that of tandem-type Usp protein Rv2623 from Mycobacterium tuberculosis. However, the dimer assembly with C2-symmetry in TTHA0350 is quite different from that with D2-symmetry in Rv2623. The X-ray structure showed that not only the N-terminal but also the C-terminal domain binds one ATP, although the ATP-binding motif could not be detected in the C-terminal domain. The loop interacting with ATP in the C-terminal domain is in a conformation quite different from that in the N-terminal domain.

Characterization of C- and N-terminal domains of Aquifex aeolicus MutL endonuclease: N-terminal domain stimulates the endonuclease activity of C-terminal domain in a zinc-dependent manner.

DNA MMR (mismatch repair) is an excision repair system that removes mismatched bases generated primarily by failure of the 3'-5' proofreading activity associated with replicative DNA polymerases. MutL proteins homologous to human PMS2 are the endonucleases that introduce the entry point of the excision reaction. Deficiency in PMS2 function is one of the major etiologies of hereditary non-polyposis colorectal cancers in humans. Although recent studies revealed that the CTD (C-terminal domain) of MutL harbours weak endonuclease activity, the regulatory mechanism of this activity remains unknown. In this paper, we characterize in detail the CTD and NTD (N-terminal domain) of aqMutL (Aquifex aeolicus MutL). On the one hand, CTD existed as a dimer in solution and showed weak DNA-binding and Mn2+-dependent endonuclease activities. On the other hand, NTD was monomeric and exhibited a relatively strong DNA-binding activity. It was also clarified that NTD promotes the endonuclease activity of CTD. NTD-mediated activation of CTD was abolished by depletion of the zinc-ion from the reaction mixture or by the substitution of the zinc-binding cysteine residue in CTD with an alanine. On the basis of these results, we propose a model for the intramolecular regulatory mechanism of MutL endonuclease activity.

Molecular mechanisms of the whole DNA repair system: a comparison of bacterial and eukaryotic systems.

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.

Structure of dihydrodipicolinate synthase from Methanocaldococcus jannaschii.

In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-beta-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2 A resolution. The structure revealed that MjDHDPS forms a functional homotetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures.

Crystallization screening test for the whole-cell project on Thermus thermophilus HB8.

It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.

Crystal structure of the YdjC-family protein TTHB029 from Thermus thermophilus HB8: structural relationship with peptidoglycan N-acetylglucosamine deacetylase.

The YdjC-family protein is widely distributed, from human to bacteria, but so far no three-dimensional structure and functional analysis of this family of proteins has been reported. We determined the three-dimensional structure of the YdjC homolog TTHB029 at a resolution of 2.9A. The overall structure of the monomer consists of (betaalpha)-barrel fold forming a homodimer. Asp21, His60, and His127 residues coordinate to Mg(2+) as a possible active site. TTHB029 shows structural similarity to the peptidoglycan N-acetylglucosamine deacetylase from Streptococcus pneumoniae (SpPgdA). The active site groove of SpPgdA includes the Zn(2+) coordinated to Asp276, His326, and His330. Despite the low sequence identity, metal-binding residues of Asp-His-His were conserved among the two enzymes. There were definitive differences, however, in that one of the histidines of the metal-binding site was substituted for the other histidine located on the other loop. Moreover, these important metal-binding residues and the residues of the presumed active site are fully conserved in YdjC-family protein.

[Feasibility of long-term outcome prediction in acute myocardial infarction using the discordance between early and delayed image on 123I-BMIPP myocardial scintigraphy].

OBJECTIVES: The feasibility of long-term outcome prediction using BMIPP myocardial scintigraphy was evaluated in cases of acute myocardial infarction. METHODS: BMIPP myocardial scintigraphy was performed on 165 patients with first acute myocardial infarction at the time of discharge from the hospital (average of 27 days after disease on set). Discordance between early and delayed image was checked and its relation to later cardiac events (during the mean follow up period of 64.2 +/- 9.8 months) was analyzed. In 82 of these 165 cases TlCl scintigraphy was simultaneously performed (Tl/BMIPP dual SPECT) to examine mismatch form BMIPP scintigraphy and discordance between early and images. RESULTS: Discordance between early and delayed images was observed in 86 cases (52%). Among patients for whom dual SPECT was performed, mismatch between TlCl and BMIPP scintigraphy was observed in 30 cases (37%). When the relation between mismatch and discordance was analyzed, mismatch was accompanied by washout. The incidence of later cardiac events was significantly higher for cases showing discordance accompanied by washout and cases showing mismatch on dual SPECT scintigraphy than cases without these findings. When multivariate analysis was conducted, involving age, sex, infarction related artery, left ventricular end-diastolic volume index, left ventricular ejection fraction, severity of disturbed fatty acid metabolism, washout and fill-in, washout was identified as an independent predictor of cardiac events. CONCLUSION: Mismatch on Tl/BMIPP dual SPECT is important for predicting long-term prognosis of acute myocardial infarction. Furthermore, washout on BMIPP scintigraphy is also useful as a predictor of cardiac events.

Structure of a conserved hypothetical protein, TTHA0849 from Thermus thermophilus HB8, at 2.4 A resolution: a putative member of the StAR-related lipid-transfer (START) domain superfamily.

The crystal structure of a conserved hypothetical protein, TTHA0849 from Thermus thermophilus HB8, has been determined at 2.4 A resolution as a part of a structural and functional genomics project on T. thermophilus HB8. The main-chain folding shows a compact alpha+beta motif, forming a hydrophobic cavity in the molecule. A structural similarity search reveals that it resembles those steroidogenic acute regulatory proteins that contain the lipid-transfer (START) domain, even though TTHA0849 shows comparatively weak sequence identity to polyketide cyclases. However, the size of the ligand-binding cavity is distinctly smaller than other START domain-containing proteins, suggesting that it catalyses the transfer of smaller ligand molecules.

[Assessment of therapeutic effect in acute myocardial infarction using early/delayed images of 123I-BMIPP myocardial scintigraphy].

This study was aimed at analyzing the discordance between the initial and late scintigraphic images in patients with acute myocardial infarction (AMI), and utilizing the data obtained for the treatment of AMI patients. Ninety-one patients with a history of the first episode of AMI were enrolled as subjects for this study. Emergency coronary angiography was performed in all the patients and left ventriculography (LVG) was carried out subsequently. 123I-BMIPP myocardial scintigraphy was performed to obtain initial images (BMi) and delayed images at 4 hours (BMd). Scintigraphy was performed a mean of 6 days after the onset of AMI in the patients. The subjects were classified into three groups according to the scintigraphic data. Quantitative gated single photon emission computed tomography (SPECT) with 99mTc-sestamibi (MIBI) was also conducted one month and 6 months later in all the patients. Discordance was observed in 51% of the patients. Left ventricular volume based on the quantitative gated SPECT (QGS) data at one month and 6 months after myocardial scintigraphy was significantly smaller in the washout group than in the other two groups. There was no significant change in LV volume measured at 6 months as compared to that measured at one month in the washout group. Significant increases in LVEDVI and LVESVI were observed over time in the no discordance group. In the fill-in group, the LV volume at one month was significantly higher than that in the washout group, but no significant change with time was observed. During the subacute stage of myocardial infarction, discordance is often seen between initial and late BMIPP-myocardial-scintigraphic images. The presence of such discordance, and analysis of its pattern, may be useful in predicting the cardiac function in these patients during the chronic phase of this disease.

Correlation between thallium-201 myocardial perfusion defects and the functional severity of coronary artery stenosis as assessed by pressure-derived myocardial fractional flow reserve.

Although a relationship between the coronary pressure-derived fractional flow reserve (FFR) and the presence of myocardial ischemia as demonstrated by radionuclide imaging has been reported in a select group of patients, it remains to be established whether this relation also holds true in actual clinical settings with a heterogeneous group of patients. Accordingly, 194 coronary vessels and their supply territories were evaluated in 165 consecutive patients with suspected or known coronary artery disease. An FFR <0.75, which is regarded as indicative of functionally important stenosis, showed a significant correlation with the redistribution of (201)Tl (p<0.0001), with a sensitivity of 79% and specificity of 73%. In 70 infarct-related coronary arteries, the sensitivity and specificity were 79% and 75%, respectively, whereas in the 124 remaining vessels that were not related to the myocardial infarct, the sensitivity and specificity were 80% and 72%, respectively. In addition, the FFR exhibited a significant inverse correlation with the (201)Tl reversibility score (r=-0.62; p<0.0001). These results suggest that the FFR has a significant relationship with scintigraphic evidence of myocardial ischemia and can be regarded as a marker of its presence or absence in patients in actual clinical settings.

High-tension electrical injury to the heart as assessed by radionuclide imaging.

To evaluate cardiac complications associated with electrical injury, 7 patients with high-tension electrical injury (6,600 V alternating current) underwent 201Tl and 123I-metaiodobenzylguanidine (MIBG) imaging in addition to conventional electrocardiographic and echocardiographic assessments. Electrocardiography showed transient atrial fibrillation, second degree atrioventricular block, ST-segment depression, and sinus bradycardia in each patient. Echocardiography showed mild hypokinesis of the anterior wall in only 2 patients, but 201Tl and 123I-MIBG myocardial scintigraphy showed an abnormal scan image in 6/7 and 5/6 patients, respectively. Decreased radionuclide accumulation was seen primarily in areas extending from the anterior wall to the septum. Decreased radionuclide accumulation was smaller in extent and milder in degree in 123I. MIBG than in 201Tl imaging. These results suggest that even in patients without definite evidence of severe cardiac complications in conventional examinations, radionuclide imaging detects significant damage due to high-tension electrical injury, in which sympathetic nerve dysfunction might be milder than myocardial cell damage.