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1.
Bozonnet S Jensen MT Nielsen MM Aghajari N Jensen MH Kramhøft B Willemoës M Tranier S Haser R Svensson B 《The FEBS journal》2007,274(19):5055-5067
Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates. 相似文献
2.
Probing the role of divalent metal ions in a bacterial psychrophilic metalloprotease: binding studies of an enzyme in the crystalline state by x-ray crystallography
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The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites. Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator. Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme. Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted approximately 4, 1.0, and 1.6 A, respectively. Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions. A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions. 相似文献
3.
Gila Pirzad Jahromi Mostafa Ghanei Seyed Kazem Hosseini Alireza Shamsaei Mazaher Gholipourmalekabadi Ameneh Koochaki Nushin Karkuki Osguei Ali Samadikuchaksaraei 《PloS one》2015,10(12)
Purpose
In patients with short-term exposure to the sulfur mustard gas, the delayed cellular effects on lungs have not been well understood yet. The lung pathology shows a dominant feature consistent with obliterative bronchiolitis, in which fibroblasts play a central role. This study aims to characterize alterations to lung fibroblasts, at the cellular level, in patients with delayed respiratory complications after short-term exposure to the sulfur mustard gas.Methods
Fibroblasts were isolated from the transbronchial biopsies of patients with documented history of exposure to single high-dose sulfur mustard during 1985–7 and compared with the fibroblasts of control subjects.Results
Compared with controls, patients’ fibroblasts were thinner and shorter, and showed a higher population doubling level, migration capacity and number of filopodia. Sulfur mustard decreased the in vitro viability of fibroblasts and increased their sensitivity to induction of apoptosis, but did not change the rate of spontaneous apoptosis. In addition, higher expression of alpha smooth muscle actin showed that the lung''s microenvironment in these patients is permissive for myofibroblastic differentiation.Conclusions
These findings suggest that in patients under the study, the delayed pulmonary complications of sulfur mustard should be considered as a unique pathology, which might need a specific management by manipulation of cellular components. 相似文献4.
Matar-Merheb R Rhimi M Leydier A Huché F Galián C Desuzinges-Mandon E Ficheux D Flot D Aghajari N Kahn R Di Pietro A Jault JM Coleman AW Falson P 《PloS one》2011,6(3):e18036
Background
Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.Methodology/Principal Findings
Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.Conclusion/Significance
These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field. 相似文献5.
Violot S Aghajari N Czjzek M Feller G Sonan GK Gouet P Gerday C Haser R Receveur-Bréchot V 《Journal of molecular biology》2005,348(5):1211-1224
Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of algae, into an immediate nutritive form by biodegrading this biopolymer. To understand the mechanisms of cold adaptation of its enzymatic components, we studied the structural properties of an endoglucanase, Cel5G, by complementary methods, X-ray crystallography and small angle X-ray scattering. Using X-ray crystallography, we determined the structure of the catalytic core module of this family 5 endoglucanase, at 1.4A resolution in its native form and at 1.6A in the cellobiose-bound form. The catalytic module of Cel5G presents the (beta/alpha)(8)-barrel structure typical of clan GH-A of glycoside hydrolase families. The structural comparison of the catalytic core of Cel5G with the mesophilic catalytic core of Cel5A from Erwinia chrysanthemi revealed modifications at the atomic level leading to higher flexibility and thermolability, which might account for the higher activity of Cel5G at low temperatures. Using small angle X-ray scattering we further explored the structure at the entire enzyme level. We analyzed the dimensions, shape, and conformation of Cel5G full length in solution and especially of the linker between the catalytic module and the cellulose-binding module. The results showed that the linker is unstructured, and unusually long and flexible, a peculiarity that distinguishes it from its mesophilic counterpart. Loops formed at the base by disulfide bridges presumably add constraints to stabilize the most extended conformations. These results suggest that the linker plays a major role in cold adaptation of this psychrophilic enzyme, allowing steric optimization of substrate accessibility. 相似文献
6.
Eun-Seong Seo Camilla Christiansen Maher Abou Hachem Morten M. Nielsen Kenji Fukuda Sophie Bozonnet Andreas Blennow Nushin Aghajari Richard Haser Birte Svensson 《Biologia》2008,63(6):967-979
α-Glucans in general, including starch, glycogen and their derived oligosaccharides are processed by a host of more or less closely related enzymes that represent wide diversity in structure, mechanism, specificity and biological role. Sophisticated three-dimensional structures continue to emerge hand-in-hand with the gaining of novel insight in modes of action. We are witnessing the “test of time” blending with remaining questions and new relationships for these enzymes. Information from both within and outside of ALAMY_3 Symposium will provide examples on what the family contains and outline some future directions. In 2007 a quantum leap crowned the structural biology by the glucansucrase crystal structure. This initiates the disclosure of the mystery on the organisation of the multidomain structure and the “robotics mechanism” of this group of enzymes. The central issue on architecture and domain interplay in multidomain enzymes is also relevant in connection with the recent focus on carbohydrate-binding domains as well as on surface binding sites and their long underrated potential. Other questions include, how different or similar are glycoside hydrolase families 13 and 31 and is the lid finally lifted off the disguise of the starch lyase, also belonging to family 31? Is family 57 holding back secret specificities? Will the different families be sporting new “eccentric” functions, are there new families out there, and why are crystal structures of “simple” enzymes still missing? Indeed new understanding and discovery of biological roles continuously emphasize value of the collections of enzyme models, sequences, and evolutionary trees which will also be enabling advancement in design for useful and novel applications. 相似文献
7.
Mohamed Ali Borgi Moez Rhimi Nushin Aghajari Mamdouh Ben Ali Michel Juy Richard Haser Samir Bejar 《Biologia》2009,64(5):845-851
The implication of the original alanine 63 (Ala63) and the unique cysteine 306 (Cys306) residues in the thermostability of
the Streptomyces sp. SK glucose isomerase (SKGI) were investigated by site-directed mutagenesis and homology modelling. The Cys306 to Ala
mutation within SKGI dramatically affected its thermal stability by decreasing the half-life from 80 to 15 min at 90°C while
the Ala63 to Ser replacement shifted this half-life to 65 min. The electrophoretic analysis proves that the residue Cys306
participates in oligomerization of the SKGI. Its stabilizing role is materialized by hydrogen bonds established with arginines
at positions 284 and 259, as deduced from the constructed three-dimensional model. We have also shown that the presence of
an Ala63 instead of Ser63 seems to be more suitable for enzyme thermostability by maintaining hydrophobic pocket that contributes
to the protection of the enzyme active site. 相似文献
8.
Gaelle Brien Anne-Laure Debaud Xavier Robert Lisa Oliver Marie-Claude Trescol-Biemont Nicolas Cauquil Olivier Geneste Nushin Aghajari Francois M. Vallette Richard Haser Nathalie Bonnefoy-Berard 《The Journal of biological chemistry》2009,284(44):30257-30263
Unlike other antiapoptotic members of the Bcl-2 family, Bfl-1 does not contain a well defined C-terminal transmembrane domain, and whether the C-terminal tail of Bfl-1 functions as a membrane anchor is not yet clearly established. The molecular modeling study of the full-length Bfl-1 performed within this work suggests that Bfl-1 may co-exist in two distinct conformational states: one in which its C-terminal helix α9 is inserted in the hydrophobic groove formed by the BH1–3 domains of Bfl-1 and one with its C terminus. Parallel analysis of the subcellular localization of Bfl-1 indicates that even if Bfl-1 may co-exist in two distinct conformational states, most of the endogenous protein is tightly associated with the mitochondria by its C terminus in both healthy and apoptotic peripheral blood lymphocytes as well as in malignant B cell lines. However, the helix α9 of Bfl-1, and therefore the binding of Bfl-1 to mitochondria, is not absolutely required for the antiapoptotic activity of Bfl-1. A particular feature of Bfl-1 is the amphipathic character of its C-terminal helix α9. Our data clearly indicate that this property of helix α9 is required for the anchorage of Bfl-1 to the mitochondria but also regulates the antiapoptotic function Bfl-1.Apoptosis is a highly regulated process that plays a key role in maintaining cellular homeostasis, and a delicate balance between proapoptotic and antiapoptotic regulators of apoptosis pathways ensures the proper survival of cells in a variety of tissues. Imbalance between proapoptotic and antiapoptotic proteins occurs in diseases such as cancer, where an overexpression of antiapoptotic proteins endows cells with a selective survival advantage that promotes malignancy. Bcl-2 family members are essential regulators of the intrinsic apoptotic pathway, which act at the level of mitochondria as initiators of cell death (1). This family comprises nearly 20 proteins divided into three main groups. Antiapoptotic members such as Bcl-2, Bcl-xL, Bcl-w, Bfl-1, and Mcl-1 promote cell survival, whereas proapoptotic members such as Bax and Bak function as death effectors. The life and death balance is displaced in favor of cell death by proapoptotic BH3-only proteins such as Bim, Bad, Bid, Puma, and Noxa, which interact with antiapoptotic proteins and inactivate their function (2) or directly interact with and activate the Bax-like proteins (3).Distinct subcellular localizations of antiapoptotic members have been reported correlating with the accessibility of their C-terminal tail. The C-terminal tail of the antiapoptotic proteins Bcl-2, Bcl-xL, and Bcl-w possess a hydrophobic region known to be a membrane anchor domain. Thus, Bcl-2 localizes to mitochondria as well as to the endoplasmic reticulum and nuclear membranes (4, 5, 6), and deletion of its C-terminal amino acids abrogates its targeting to the outer mitochondrial membrane (7). In contrast, in healthy cells, Bcl-xL and Bcl-w localize mainly in the cytosol because their C-terminal tails are sequestered. Bcl-xL exists as a homodimer through the exchange of the C-terminal tail bound in the hydrophobic groove of the reciprocal dimer partner (8), whereas the C-terminal tail of Bcl-w occupies its own hydrophobic groove in the monomer form (9, 10). It has been proposed that, following apoptotic stimuli, interaction of the BH3 domain from BH3-only proteins with the hydrophobic groove of Bcl-w or Bcl-xL liberates their C-terminal tail and then the two proteins translocate to the mitochondria (8, 11).Unlike Bcl-2, Bcl-xL, and Bcl-w, Bfl-1 and its murine homolog, A1, do not contain a well defined C-terminal transmembrane domain (12, 13). C-terminal ends of these two proteins are similar and contain several hydrophilic residues that interrupt their putative transmembrane hydrophobic domain. Whether the C-terminal tail of Bfl-1 functions as a membrane anchor remains to be clarified. Immunofluorescence analyses in an earlier study have shown that overexpressed human Bfl-1 is predominantly localized in the endoplasmic/nuclear envelope regions (14). Then, recent independent studies, with Bfl-1-overexpressing cells, suggested that Bfl-1 localizes to the mitochondria (15, 16, 17) and that the C-terminal end of Bfl-1 is important for anchoring Bfl-1 to the mitochondria due to GFP-Bfl-1 being associated to the mitochondria, whereas GFP-Bfl-1, devoid of its C-terminal tail, also localizes in the cytosol (16, 18). However, localization of endogenous Bfl-1 has never been investigated. In this study, we present a molecular modeling study of full-length Bfl-1 (FL-Bfl-1), based on the crystal structure of a truncated form of Bfl-1 (residues 1–149) in complex with the BIM-BH3 peptide (Protein Data Bank code 2VM6).4 Our model suggests that Bfl-1 may co-exist in two distinct conformational states, the first one with its C-terminal helix α9 (residues 155–175) inserted in the hydrophobic groove formed by the BH1–3 domain of Bfl-1, and the second one with its C-terminal tail. Interestingly, helical wheel projection of the C-terminal helix of Bfl-1 highlights its amphipathic character, a feature of transmembrane helices or membrane anchors. These observations incited the reinvestigation of the subcellular localization of Bfl-1 in both malignant B cell lines and peripheral blood lymphocytes (PBLs).5 We demonstrate here that endogenous Bfl-1 is preferentially anchored to the mitochondria in malignant B cell lines but also in healthy PBLs. Moreover, we show that both the anchorage of Bfl-1 to the mitochondria and the anti-apoptotic function of the protein are dependent on the amphipathic nature of the C-terminal helix. 相似文献
9.
The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the sucrose isomerase SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics of the isomaltulose synthases catalytic pocket and their substrate binding mode. Comparison with the trehalulose synthase MutB highlights the role of Arg298 and Arg306 active site residues and surface charges in controlling product specificity of sucrose isomerases (isomaltulose versus trehalulose). The results provide a rationale for the specific design of optimized enzymes. 相似文献
10.
Bassem Khemakhem Mamdouh Ben Ali Nushin Aghajari Richard Haser 《Biochemical and biophysical research communications》2009,385(1):78-270
To provide insight into the potential role of a loop in domain B of several bacterial α-amylases, molecular and structural investigation of Bacillus stearothermophilus α-amylase (Amy US100) was used as a model. Combination deletion mutants of G213, I214 and G215, described as a loop-forming on the surface bacterial amylases, were subjected to biochemical and structural investigation. Thermoactivity, thermostability as well calcium requirement were studied for each mutant.Thus, deletion of one residue differently affects only the thermostability. Shortening the loop by deletion of G213-I214 or I214-G215 improved the thermostability and reduces calcium requirement. However, the deletion of three residues has a negative effect on thermostability and reduces the optimal temperature by 17 °C.The structural investigation showed that stabilizing deletions contribute to reinforce the architecture of domain B and the active site conformation. The deletion of three residues reduces the flexibility of this region and abolishes a denser hydrogen bond network. 相似文献