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Background: We demonstrated that dipeptidyl-peptidase-4 inhibitor (DPP4i) Des-Fluoro-Sitagliptin (DFS) exhibited anti-atherosclerotic effects on the newly development process of atherosclerosis, however it is unknown whether DPP4i also have anti-atherosclerotic effects on the established atheroma (preventing progression or regression). In the practical situation, it is clinically important to investigate whether DPP4i have beneficial effects on the established atherosclerosis. Methods and Results: 8 weeks old ApoE deficient mice were fed by high fat diet (HFD) for 12 weeks lead to development of the established atherosclerotic plaques, followed by randomized into 4 groups; continued HFD arm [HFD alone, HFD+DFS (200mg/kg/day)] and lipid-lowering arm [low fat diet (LFD) alone, LFD+DFS], for additional 8 weeks. DFS significantly reduced the progression of established plaque compared to HFD alone [plaque area per aorta; HFD (n=11): 53.3±4.9% vs. HFD+DFS (n=15): 43.8±9.0%, p Conclution: DFS exhibited beneficial effects on the established atherosclerosis with attenuating progression and supporting regression by the lipid-lowering therapy. AMPK activation by DFS might partly play a role in these processes. DFS could also be effective for the established atherosclerosis.

Background: Inhibiting Neutral Endpeptidase-24.11 (NEP) combined with angiotensin receptor blocker (ARB) has recently been employed as an effective treatment for hypertension (HT). Although soluble form of NEP (sNEP) is able to measure in human plasma, the relationship between plasma levels of sNEP and clinical condition is still unknown in HT patients. Methods: We consecutively measured plasma levels of sNEP and B-type natriuretic peptide (BNP) in 210 patients with HT (144 male, 66 female, mean age; 69.5±10.4) who were suspected cardiovascular disease with multiple risk factors from April 2013 to December 2013. The levels of plasma sNEP were measured by using commercially available ELISA kits (SEB785Hu, USCN life science inc.). Results: Plasma levels of sNEP were non-normally distributed in whole high-risk HT patients assessed by Shapiro-Wilk test (p Conclusion: Among high risk patients with HT, patients treated with ACEI demonstrated significantly elevated levels of sNEP and the administration of ACEI was independently associated with the higher levels of sNEP. NEP could be up-regulated by ACEI and ARB did not significantly affect on NEP activity in HT patients. NEP could be effectively inhibited by a NEP-inhibitor in HT patients treated by ARB.

Background: Mitochondrial dysfunction plays an important role in cellular senescence and impaired function of vascular endothelium, resulted in cardiovascular diseases. AMP-activated protein kinase (AMPK) plays a critical role in mitochondrial biogenesis and endothelial function. Telmisartan (TELMI) is a unique angiotensin II type I receptor blocker (ARB) that has clinically been shown to prevent cardiovascular events in high risk patients. We investigated whether TELMI could modulate mitochondrial function leads to improvement of endothelial function via AMPK, and could improve endothelial function in mice and human. Methods and Results: In cultured human coronary artery endothelial cells (HCAECs), TELMI significantly increased the phosphorylation of AMPK (1.61±0.45 fold, p Conclusions: TELMI significantly enhanced mitochondrial function and exhibited anti-senescent activity via AMPK in HCAECs, and improved endothelial function in mice and human. The vascular protective effects induced by TELMI may partly be due to its enhancement of mitochondrial function and reduction of endothelial senescence by AMPK.

Polyvascular disease (PolyVD) refers to the coexistence of coronary artery disease (CAD), peripheral arterial disease (PAD), and/or cerebrovascular disease (CVD), and carries a high risk of cardiovascular mortality. Endothelial dysfunction plays a crucial role in cardiovascular pathophysiology. This study investigated the association between PolyVD and the presence of microvascular endothelial dysfunction.Consecutive stable patients (n=533) with diabetes mellitus and/or multiple cardiovascular risk factors were enrolled. Peripheral microvascular endothelial function in the finger microvasculature was assessed using the reactive hyperemia peripheral arterial tonometry index (RHI), and ankle-brachial index was measured for diagnosis of lower-extremity PAD prior to coronary angiography. Diagnosis of CVD was based on clinical symptoms, carotid ultrasound, and magnetic resonance imaging. PolyVD was defined as two or more coexisting vascular diseases from CAD, lower-extremity PAD, and CVD.Natural logarithmic transformations of RHI (Ln-RHI) were significantly attenuated in 93 patients with PolyVD (0.44±0.20) compared with those in 440 patients without PolyVD (0.56±0.19; p0.001) or in 299 patients with a single vascular disease (0.54±0.19; p0.001). There was an independent correlation between Ln-RHI (per 0.1) and the presence of PolyVD in all high-risk patients [odds ratio (OR): 0.724; 95% confidence interval (CI): 0.610-0.859; p0.001] and one or more vascular diseases (OR: 0.724; 95% CI: 0.605-0.867, p0.001). Receiver-operating characteristics curve analysis showed that Ln-RHI correlated significantly with PolyVD (area under the curve, 0.682, 95% CI: 0.625-0.740, p0.001). The optimum cut-off point of Ln-RHI for the existence of PolyVD was 0.479.Microvascular endothelial dysfunction is significantly associated with the presence of PolyVD. Severe impairment of endothelial function in peripheral microvasculature may be an important pathophysiological component of PolyVD.

Background Pentraxin 3 ( PTX 3) is a novel inflammatory marker produced by various cell types including those of the vasculature and the heart. The relationship between inflammatory markers and prognosis of patients with heart failure with normal ejection fraction ( HFNEF ) remains unknown. We investigated whether plasma PTX 3 levels can predict future cardiovascular events in patients with HFNEF . Methods and Results Plasma PTX3, high‐sensitivity C‐reactive protein, and B‐type natriuretic peptide levels were measured prospectively in 360 stable patients with HFNEF. The subsequent incidence of cardiovascular events, including cardiovascular death, nonfatal myocardial infarction (MI), unstable angina pectoris, nonfatal ischemic stroke, hospitalization for heart failure decompensation, and coronary revascularization, was determined. During a mean 30‐month follow‐up, 106 patients experienced cardiovascular events. These events were more frequent in patients with high plasma PTX3 levels (>3.0 ng/ mL ) than low levels (≤3.0 ng/ mL ). Multivariable Cox hazard analysis showed that PTX3 (hazard ratio: 1.16; 95% CI: 1.05 to 1.27; P P P Conclusions High plasma PTX 3 levels, but not other inflammatory markers, are correlated with future cardiovascular events in patients with HFNEF . PTX 3 may be a useful biomarker for assessment of risk stratification in HFNEF . Clinical Trial Registration URL : http://www.umin.ac.jp ; Unique identifier: UMIN 000002170.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by a condensation of isopentenyl diphosphate to an allylic diphosphate. A novel gene encoding a cis-prenyltransferase is cloned from Thermobifida fusca. It showed a unique substrate specificity accepting dimethylallyl diphosphate as a shortest allylic substrate, and synthesizes polyprenyl products up to C(70).

The three-dimensional crystal structure of hen apo-ovotransferrin has been solved by molecular replacement and refined by simulated annealing and restrained least squares to a 3.0-A resolution. The final model, which comprises 5312 protein atoms (residues 1 to 686) and 28 carbohydrate atoms (from two monosaccharides attached to Asn473), gives an R-factor of 0.231 for the 11,989 observed reflections between 20.0- and 3.0-A resolution. In the structure, both empty iron binding clefts are in the open conformation, lending weight to the theory that Fe3+binding or release in transferrin proceeds via a mechanism that involves domain opening and closure. Upon opening, the domains rotate essentially as rigid bodies. The two domains of the N-lobe rotate away from one another by 53°, whereas the C-lobe domains rotate away each another by 35°. These rotations take place about an axis that passes through the two β-strands, linking the domains. The domains of each lobe make different contacts with one another in the open and closed forms. These contacts form two interdomain interfaces on either side of the rotation axis, and domain opening or closing produces a see-saw motion between these two alternative close-packed interfaces. The interdomain disulfide bridge (Cys478-Cys671), found only in the C-lobe, may restrict domain opening but does not completely prevent it.

Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO3 2−. Upon Fe3+uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 A resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121.

Background Endothelial dysfunction is a key component of vascular vulnerability. Reactive hyperemia index ( RHI ), as assessed by the peripheral arterial tonometry, can noninvasively evaluate endothelial function. This study was designed to determine the additional prognostic value of endothelial function to the Synergy Between PCI With Taxus and Cardiac Surgery Score ( SYNTAX sc) and the Framingham Risk Score ( FRS ) in predicting cardiovascular events in high‐risk patients. Methods and Results We undertook a two‐center prospective study in 528 stable patients at high‐risk for cardiovascular events from the years 2006–2011. The RHI was measured before coronary angiography and coronary complexity was assessed by SYNTAX sc. After optimal therapies including coronary revascularization, there was follow‐up with patients until August 2012. Cardiovascular events consist of cardiovascular death, myocardial infarction, unstable angina, ischemic stroke, coronary revascularization, heart failure‐induced hospitalization, aortic disease, and peripheral arterial disease. During 1468 person‐years of follow‐up, 105 patients developed cardiovascular events. Multivariate Cox proportional hazards analysis identified B‐type natriuretic peptide ( BNP ), SYNTAX sc, and RHI as independent cardiovascular event predictors (hazard ratio [95% confidence interval]: natural logarithm of BNP per 0.1: 1.019 [1.002 to 1.037]; P =0.023, SYNTAX sc per tertile: 2.426 [1.825 to 3.225]; P RHI per 0.1: 0.761 [0.673 to 0.859]; P RHI was added to the FRS , BNP , and SYNTAX sc, net reclassification index was significantly improved (27.5%; P P =0.031). Conclusions Advanced endothelial dysfunction significantly correlated with near future cardiovascular events in high‐risk patients. This physiological vascular measurement improved risk discrimination when added to the FRS , BNP , and SYNTAX sc. Clinical Trial Registration URL : clinicaltrials.gov ( http://www.clinicaltrials.gov ). Unique identifier: NCT 00737945.

We thank Drs Emanuele Barbato and Fabio Mangiacapra for their careful and insightful comments on our article1 demonstrating the correlation between peripheral endothelial function assessed by reactive hyperemia index (RHI) and residual platelet reactivity (RPR). As they pointed out, in our population, there was a higher rate of patients with multivessel disease (MVD) in the high RPR group. This might be because it was a selected population without CYP2C19*2 or *3 loss-of-function alleles. We could not demonstrate the correlation between RPR and the severity and extent of coronary artery disease (CAD) because the sample size was limited. However, we …

Background Angina without significant stenosis, or nonobstructive coronary artery disease, attracts clinical attention. Microvascular coronary artery spasm (microvascular CAS ) can cause nonobstructive coronary artery disease. We investigated the clinical features of microvascular CAS and the therapeutic efficacy of calcium channel blockers. Methods and Results Three hundred seventy consecutive, stable patients with suspected angina presenting nonobstructive coronary arteries (CAS according to lactate production and a decrease in coronary blood flow without epicardial vasospasm during the acetylcholine provocation test. We prospectively followed up the patients with calcium channel blockers for microvascular coronary artery disease. We identified 50 patients with microvascular CAS who demonstrated significant impairment of the endothelium‐dependent vascular response, which was assessed by coronary blood flow during the acetylcholine provocation test. Administration of isosorbide dinitrate normalized the abnormal coronary flow pattern in the patients with microvascular CAS . Multivariate logistic regression analysis indicated that female sex, a lower body mass index, minor–borderline ischemic electrocardiogram findings at rest, limited–baseline diastolic‐to‐systolic velocity ratio, and attenuated adenosine triphosphate–induced coronary flow reserve were independently correlated with the presence of microvascular CAS . Receiver‐operating characteristics curve analysis revealed that the aforementioned 5‐variable model showed good correlation with the presence of microvascular CAS (area under the curve: 0.820). No patients with microvascular CAS treated with calcium channel blockers developed cardiovascular events over 47.8±27.5 months. Conclusions Microvascular CAS causes distinctive clinical features and endothelial dysfunction that are important to recognize as nonobstructive coronary artery disease so that optimal care with calcium channel blockers can be provided. Clinical Trial Registration URL: www.umin.ac.jp/ctr . Unique identifier: UMIN000003839.

Prenyltransferases catalyze the fundamental isoprenoid chain-elongation process to produce prenyl diphosphates with various chain lengths and stereochemistries. The reactions are regulated to proceed consecutively and terminate precisely at definite chain lengths according to the specificities of individual enzymes. Comparison of many prenyltransferases revealed that prenyltransferases are classified into two groups, trans- or (E)-prenyltransferases and cis- or (Z)-prenyltransferases. Molecular mechanisms of the catalytic functions of trans- and cis-prenyltransferases based on the recently accumulating reports accomplished by crystallographic structural studies of prenyltransferases as well as the chain-length regulation mechanism based on the site-directed mutagenesis studies recently carried out are introduced.

Maf transcription factors constitute a family of the basic region-leucine zipper (bZip) factors and recognize unusually long DNA motifs (13 or 14 bp), termed the Maf recognition element (MARE). The MARE harbors extended GC sequences on each side of its core motif, which is similar to TRE or CRE (7 or 8 bp) recognized by the AP1 and CREB/ATF families, respectively. To ascertain the structural basis governing the acquirement of such unique DNA recognition, we determined the crystal structure of the MafG-DNA complex. Each MafG monomer consists of three helices in which the carboxyl-terminal long helix organizes one DNA-contacting element and one coiled-coil dimer formation element. To our surprise, two well-conserved residues, Arg57 and Asn61 in the basic region, play critical roles in Maf-specific DNA recognition. These two residues show unique side-chain orientations and interact directly with the extended GC bases. Maf-specific residues in the amino-terminal and basic regions appear to indirectly stabilize MARE recognition through DNA backbone phosphate interactions. This study revealed an alternative DNA recognition mechanism of the bZip factors that bestows specific target gene profiles upon Maf homodimers or Maf-containing heterodimers.

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by sequential condensation of isopentenyl diphosphate with allylic diphosphate. cis-Prenyltransferases can be classified into three subgroups: short-, medium-, and long-chain cis-prenyltransferase, according to their product chain lengths. cis-Farnesyl diphosphate synthase from Mycobacterium tuberculosis has been the only example as short-chain cis-prenyltransferase so far characterized. In this study, we cloned the novel short-chain cis-prenyltransferases from three different bacteria, and characterized their enzymatic activities to compare and elucidate a common feature of the short-chain cis-prenyltransferases. Furthermore, we identified a specific isoleucine that is conserved in short-chain cis-prenyltransferases and located in close proximity of the omega-end of the geranyl diphosphate. Several site-directed mutants with respect to the isoleucine residue synthesized longer prenyl chain products and showed broader allylic substrate specificity. These results suggested that the isoleucine plays an important role in the substrate specificity and chain length determination mechanism of cis-prenyltransferase.

In the small Q{sup 2} region, the sum rule for the polarized structure function g{sub 2} corresponding to the moment at n=0 is derived. This sum rule shows that there is a tight connection among the resonances, the elastic and the continuum in the g{sub 2}. Further, the Born term contribution in this sum rule is proportional to Q{sup 2} and very small compared with that in the corresponding sum rule for the polarized structure function g{sub 1}. However, the Born term contribution divided by Q{sup 2}/2 which also appears in the Schwinger sum rule for the g{sub 2} corresponding to the moment at n=1 has a very similar behavior with that in the sum rule for the g{sub 1} corresponding to the moment at n=0.

Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated transcription factor with two heme-binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme-binding kinetics and DNA-binding characteristics of the isolated fragment containing the N-terminal basic helix-loop-helix (bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the PAS-A domain alone. We found that the heme-bound bHLH-PAS-A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS-A (412 nm). The axial ligand trans to CO in bHLH-PAS-A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo-bHLH-PAS (3.3 x 10(7) mol(-1) x s(-1)) was more than two orders of magnitude higher than for association with apo-PAS-A (10(5) mol(-1) x s(-1)). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)-NO heme complex is five-coordinated. Using the quartz-crystal microbalance method, we found that the bHLH-PAS-A domain binds specifically to the E-box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH-PAS-A and its potential role in regulating DNA binding.

Recent studies have revealed a new class of heme enzymes, the heme-based sensors, which are able to turn on or off cellular signal transduction pathways in response to environmental changes. One of these enzymes is the heme-regulated phosphodiesterase from Escherichia coli (EcDOS). This protein is composed of an N-terminal heme-containing PAS domain and a C-terminal functional domain. PAS is an acronym formed from the names of the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM). The heme cofactor in its PAS domain can act as a sensor of the cellular redox state that regulates the adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase activity. The crystal structures of its heme-containing PAS domain have helped clarify how the heme redox-dependent structural changes initiate intramolecular signal transduction. Here, we review recent findings on the structure-function relationships of EcDOS.

Neuronal PAS domain protein 2 (NPAS2) is an important transcription factor associated with circadian rhythms. This protein forms a heterodimer with BMAL1, which binds to the E-box sequence to mediate circadian rhythm-regulated transcription. NPAS2 has two PAS domains with heme-binding sites in the N-terminal portion. In this study, we overexpressed wild-type and His mutants of the PAS-B domain (residues 241-416) of mouse NPAS2 and then purified and characterized the isolated heme-bound proteins. Optical absorption spectra of the wild-type protein showed that the Fe(III), Fe(II) and Fe(II)-CO complexes are 6-co-ordinated low-spin complexes. On the other hand, resonance Raman spectra indicated that both the Fe(III) and Fe(II) complexes contain mixtures of 5-co-ordinated high-spin and 6-co-ordinated low-spin complexes. Based on inverse correlation between nu(Fe-CO) and nu(C-O) of the resonance Raman spectra, it appeared that the axial ligand trans to CO of the heme-bound PAS-B is His. Six His mutants (His266Ala, His289Ala, His300Ala, His302Ala, His329Ala, and His335Ala) were generated, and their optical absorption spectra were compared. The spectrum of the His335Ala mutant indicated that its Fe(III) complex is the 5-co-ordinated high-spin complex, whereas, like the wild-type, the complexes for the five other His mutants were 6-co-ordinated low-spin complexes. Thus, our results suggest that one of the axial ligands of Fe(III) in PAS-B is His335. Also, binding kinetics suggest that heme binding to the PAS-B domain of NPAS2 is relatively weak compared with that of sperm whale myoglobin.

In haem-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS), haem is bound to the PAS domain, and the redox state of the haem iron regulates catalysis by the PDE domain. We generated mutants of Asp40, which forms a hydrogen bond with His77 (a proximal haem axial ligand) via two water molecules, and a salt bridge with Arg85 at the protein surface. The redox potential of haem was markedly increased from 67 mV vs. the standard hydrogen electrode in the wild-type enzyme to 95 mV and 114 mV in the Ala and Asn mutants, respectively. Additionally, the auto-oxidation rate of Ec DOS PAS was significantly increased from 0.0053 to 0.051 and 0.033 min(-1), respectively. Interestingly, the catalytic activities of the Asp40 mutants were abolished completely. Thus, Asp40 appears to play a critical role in the electronic structure of the haem iron and redox-dependent catalytic control of the PDE domain. In this report, we discuss the mechanism of catalytic control of Ec DOS, based on the physico-chemical characteristics of the Asp40 mutants.

Publisher Summary The histidine kinase protein, the principal component of protein phosphotransfer in bacteria, plays a central role in the signaling pathways required for the environmental adaptation of these organisms. Histidine kinases are composed of a central dimerization domain and an ATP-binding domain (which is highly conserved among members of this protein family, but distinct in primary sequence from Ser/Thr/Tyr protein kinases) and other structural modules such as the sensor domain and the phosphotransfer domain. Advances in three-dimensional structural studies of these domains, or the essential building blocks, from the various histidine kinases have deciphered both a functional and an evolutionary link between the members of this protein family as well as between these and other proteins. Most notably, the α/β sandwich fold identified in both EnvZ and CheA ATP-binding domains revealed a marked resemblance to the fold found in the GHL ATPase family containing Hsp90, DNA gyrase B, and MutL. This chapter discusses the structurally characterized building blocks that are essential for the activity and regulation of histidine kinases.

Histidine kinases function as dimers. The kinase domain of the osmosensing histidine kinase EnvZ of Escherichia coli consists of two domains: domain A (67 residues) responsible for histidine phosphotransfer and dimerization, and domain B (161 residues) responsible for the catalytic and ATP-binding function. The individual structures of these two domains have been recently solved by NMR spectroscopy. Here, we demonstrate that an enzymatically functional monomeric histidine kinase can be constructed by fusing in tandem two domains A and one domain B to produce a single polypeptide (A-A-B). We show that this protein, EnvZc[AAB], is soluble and exists as a stable monomer. The autophosphorylation and OmpR kinase activities of the monomeric EnvZc[AAB] are similar to that of the wild-type EnvZ, while OmpR-binding and phosphatase functions are reduced. V8 protease digestion and mutational analyses indicate that His-243 of only the amino proximal domain A is phosphorylated. Based on these results, molecular models are proposed for the structures of EnvZc[AAB] and the kinase domain of EnvZ. The present results demonstrate for the first time the construction of a functional, monomeric histidine kinase, further structural studies of which may provide important insights into the structure-function relationships of histidine kinases.

Following the multiplication of biochemical, biophysical and structural studies describing cadherin molecules and their interactions, several ideas have emerged to explain the mechanisms of cadherin-mediated cell adhesion. Although different models were proposed for cadherin interactions, a consensus has come forth considering lateral dimerization of cadherins as being a central component of the cell-cell adhesion process. This review summarizes the recent development in structural studies of cadherins.

Derivatives of pterin, an important cofactor for neuronal nitric oxide synthase, markedly changed the kinetics of CO binding to the enzyme-bound heme. In the absence of the substrate, L-Arg, CO bin...

Fluorescence bands of a Trp110Ile mutant of the isolated heme domain of a heme-regulated phosphodiesterase (Ec DOS) from Escherichia coli and its complex with 8-anilino-1-naphthalenesulfonic acid (...

The three-dimensional structure of diferric hen ovotransferrin has been determined by X-ray crystallography at 2.4 A resolution. The structure was solved by molecular replacement, using the coordinates of diferric human lactoferrin as a search model. Several rounds of simulated annealing and restrained least-squares refinement have resulted in a model structure with an R -factor of 0.171 for the data between 11.0 and 2.4 A resolution. The model comprises 5284 protein atoms (residues 5 to 686), 2 Fe 3+ , 2 CO 3 2− and 132 water molecules. The overall structure of ovotransferrin is similar to those of human lactoferrin and rabbit serum transferrin, being folded into two homologous lobes, each containing two dissimilar domains with one F 3+ e and one CO 2− 3 bound at a specific site in each interdomain cleft. However, the relative orientation of the two lobes, which may be related to the class specificity of transferrins to receptors, is different from either human lactoferrin or rabbit serum transferrin. The angle of the relative orientation in ovotransferrin is increased by 6.8° and 15.7° as compared with to those in rabbit serum transferrin and human lactoferrin, respectively. Interdomain Lys209-Lys301 and Gln541-Lys638 interactions are found near the metal binding site of each lobe. The interlobe interactions and their role in the stabilization of iron binding are discussed.

Abstract 3441 Transcription factor GATA1 regulates a set of genes essential for the erythroid and megakaryocytic cell differentiation through the interaction with GATA motifs (consensus sequence: A/TGATAA/T). Two zinc fingers within GATA1 have been identified to be important in the DNA binding of GATA1, which are referred to as C-finger (CF) and N-finger (NF) domains. It has been shown that transactivation activity of GATA1 is completely abolished upon deletion of the CF domain, indicating that the CF domain is a requisite for the DNA binding of GATA1. While conventional reporter transactivation analyses hardly clarified the importance of the NF domain for the DNA binding, substitution mutations on 216th arginine (R216) located in the DNA-interacting surface of the NF domain have been identified to cause familial diseases of thrombocytopenia, thalassemia, and porphyria. As a consequence of the substitution of R216 to glutamine (Q) or tryptophan (W), DNA binding activity of GATA1 to a palindromic configuration of two GATA motifs (palindromic GATA) was largely diminished, while that to a single GATA motif was maintained. In this study we have examined the DNA binding diversity of GATA1 caused by the difference in the configuration of GATA motifs. We performed surface plasmon resonance (SPR) analyses of GATA1 to a single GATA, a palindromic GATA, and a repeating configuration of two GATA motifs (tandem GATA). We found that GATA1 binds to the palindromic GATA motif in a bivalent way, while it binds to the single GATA motif in a monovalent mode. We also found that a double quantity of GATA1 is associated with the tandem GATA motif and GATA1 lacking the NF domain binds to any configurations of GATA motif in a monovalent way. To further investigate contribution of the NF domain to the binding mode of GATA1, we have constructed two types of GATA1 mutants; one type was the substitution mutations on R216 (R216Q and R216W) that were mouse homologues of the human mutations, while the other type was the alanine substitution mutation on three lysine residues (K245, K246 and K312; referred to as 3KA mutant), whereby dimerization potential of GATA1 was reduced to trace level similar to the case for GATA1 lacking the NF domain. Impotantly, R216Q and R216W mutants bind the palindromic GATA motif in a monovalent way, while these mutants bind normally to the other configuration of GATA motifs. In contrast, we found that one molecule of 3KA mutant bound to the tandem GATA motif and this observation seems to explain well the fact that dimerization potential of GATA1 is an important requisite for the full-function of GATA1 in embryos. The binding modes of this 3KA mutant to the other configurations were not influenced. These results thus demonstrate that the both NF and CF domains recognize the multiple configurations of GATA motifs and specify the binding modes of GATA1. Importantly, GATA1-deficient mice rescued with R216Q were lethal during late gestation period due to abnormality in erythroid differentiation, indicating that the contribution of the NF domain to the recognition of the palindromic GATA motif configuration indeed functions in vivo. These results thus support our contention that the NF domain acts to regulate a proper spatio-temporal gene expression of a subset of GATA1 target genes utilizing the variations in the GATA motif configuration. Disclosures: No relevant conflicts of interest to declare.

The mechanism for reversible iron binding in the N-terminal lobe of ovotransferrin was investigated by a protein fragmentation approach. The iron-saturated N-terminal half-molecule of ovotransferrin was proteolyzed into large 30-kDa (1-279) and small 6-kDa (280-332) fragments by a single cleavage with Achromobacter protease I, producing a stable nicked form. For the separation of the two fragments, denaturing conditions were required. The isolated large fragment contained all four iron-coordinating ligands and the anion-binding ligand, and according to spectroscopic titration analysis, it showed iron binding capacity. The iron-bound large fragment, however, showed a blue-shifted visible absorption spectrum and a much decreased iron stability compared with those of the intact half-molecule. Analyses by ion-exchange chromatography revealed that the large fragment reassociates with the small fragment. In order for reassociation to occur, iron must be bound to the large fragment; upon reassociation, the large fragment regained its stable iron binding capacity as well as a visible absorption spectrum almost indistinguishable from those of the intact half-molecule. These data are consistent with a two-step sequential pathway for reversible iron binding in the N-terminal lobe of ovotransferrin that includes an initial iron binding intermediate having a perturbed metal environment and its transformation into the stable iron-bound holoform by peptide-peptide interactions between the large coordinating and small noncoordinating segments.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone and has cardiovascular protective effects [(1)][1]. Vascular cells express GLP-1 receptors, and studies have demonstrated that enhancing GLP-1 activity exhibits beneficial effects on atherosclerosis in animal models [(2)][2]. However, the impact