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Loop Regions (loop + regions)
Selected AbstractsThe crystal structure of pyruvate decarboxylase from Kluyveromyces lactisFEBS JOURNAL, Issue 18 2006Implications for the substrate activation mechanism of this enzyme The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 Å resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106,113 and 292,301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105,113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105,113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase. [source] Probing the unfolding region of ribonuclease A by site-directed mutagenesisFEBS JOURNAL, Issue 20 2004Jens Köditz Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent ,-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in ,G can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule. [source] Effects of proline mutations in the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activityFEBS JOURNAL, Issue 22 2000Toshiro Takai Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino-acid residues. Wild-type and six proline mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed in Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The apparent molecular masses analyzed by gel-filtration were 14,15 kDa. The IgE-binding capacity in the sera of seven mite-allergic patients, inhibitory activity for IgE-binding to immobilized wild-type Der f 2, and activity to stimulate peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild-type Der f 2 in their CD spectrum, showed reduced IgE-binding, reduced inhibitory activity, and less histamine-releasing activity than the wild-type. P34A also showed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are closely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE-binding site common to all tested patients. P66A showed reduced IgE-binding in two sera out of seven. P26A and P79A showed no reduced allergenicity. However, in immunoblot analysis after SDS/PAGE under reduced conditions, P79A showed no or markedly reduced IgE-binding while the other mutants showed IgE-binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy. [source] Scanning mutagenesis of regions in the G, protein Gpa1 that are predicted to interact with yeast mating pheromone receptorsFEMS YEAST RESEARCH, Issue 1 2008Douglas P. Gladue Abstract The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive G, protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of G, are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (,2,,3, ,2,,4, ,3,,5, and ,4,,6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by G,,. However, the constitutive activity caused by the F344C and E335C mutations in the ,2,,4 loop and F378C in the ,3,,5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering G,,. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the ,2,,3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of G, contribute to activation of signaling. [source] The two-hydrophobic domain tertiary structure of reticulon proteins is critical for modulation of ,-secretase BACE1JOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2009Hideaki Kume Abstract ,-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a membrane-bound protease that is essential for the production of ,-amyloid protein (A,). Given the crucial role of A, accumulation in Alzheimer's disease (AD), inhibition of BACE1 activity may represent a feasible therapeutic strategy in the treatment of AD. Recently, we and others identified reticulon 3 (RTN3) and reticulon 4-B/C (RTN4-B/C or Nogo-B/C) as membrane proteins that interact with BACE1 and inhibit its ability to produce A,. In this study, we employed various mutants of RTN3 and RTN4-C and C. elegans RTN to investigate the molecular mechanisms by which RTNs regulate BACE1. We found that RTN3 mutants lacking the N-terminal or C-terminal or loop domain as well as a RTN4-C mutant lacking the C-terminal domain bound to BACE1 comparably to wild-type RTN3 and RTN4-C. Furthermore, overexpression of wild-type RTN3, RTN4-C, and these RTN mutants similarly reduced A,40 and A,42 secretion by cells expressing Swedish mutant APP. C. elegans RTN, which has low homology to human RTNs, also interacted with BACE1 and inhibited A, secretion. In contrast, two RTN3 mutants containing deletions of the first or second potential transmembrane domains and an RTN3 swap mutant of the second transmembrane domain bound BACE1 but failed to inhibit A, secretion. Collectively, these results suggest that the two-transmembrane-domain tertiary structure of RTN proteins is critical for the ability of RTNs to modulate BACE1 activity, whereas N-terminal, C-terminal and loop regions are not essential for this function. © 2009 Wiley-Liss, Inc. [source] Crystal structure of enoyl,acyl carrier protein reductase (FabK) from Streptococcus pneumoniae reveals the binding mode of an inhibitorPROTEIN SCIENCE, Issue 4 2008Jun Saito Abstract Enoyl,acyl carrier protein (ACP) reductases are critical for bacterial type II fatty acid biosynthesis and thus are attractive targets for developing novel antibiotics. We determined the crystal structure of enoyl,ACP reductase (FabK) from Streptococcus pneumoniae at 1.7 Å resolution. There was one dimer per asymmetric unit. Each subunit formed a triose phosphate isomerase (TIM) barrel structure, and flavin mononucleotide (FMN) was bound as a cofactor in the active site. The overall structure was similar to the enoyl,ACP reductase (ER) of fungal fatty acid synthase and to 2-nitropropane dioxygenase (2-ND) from Pseudomonas aeruginosa, although there were some differences among these structures. We determined the crystal structure of FabK in complex with a phenylimidazole derivative inhibitor to envision the binding site interactions. The crystal structure reveals that the inhibitor binds to a hydrophobic pocket in the active site of FabK, and this is accompanied by induced-fit movements of two loop regions. The thiazole ring and part of the ureido moiety of the inhibitor are involved in a face-to-face ,,, stacking interaction with the isoalloxazine ring of FMN. The side-chain conformation of the proposed catalytic residue, His144, changes upon complex formation. Lineweaver,Burk plots indicate that the inhibitor binds competitively with respect to NADH, and uncompetitively with respect to crotonoyl coenzyme A. We propose that the primary basis of the inhibitory activity is competition with NADH for binding to FabK, which is the first step of the two-step ping-pong catalytic mechanism. [source] All three Ca2+ -binding loops of photoproteins bind calcium ions: The crystal structures of calcium-loaded apo-aequorin and apo-obelinPROTEIN SCIENCE, Issue 3 2005Lu Deng HLH, helix,loop,helix; HSQC, heteronuclear single quantum coherence; RMSD, root mean square deviation; SAD, single wavelength anomalous dispersion Abstract The crystal structures of calcium-loaded apoaequorin and apo-obelin have been determined at resolutions 1.7 Å and 2.2 Å, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-proteins retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hydroperoxycoelenterazine, and also the same as the Ca2+ -discharged obelin bound with the product, coelenteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these subtle shifts are the basis of the ability of these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed. [source] Purification, crystallization and preliminary X-ray analysis of the GTP-binding protein Rab9 implicated in endosome-to-TGN vesicle traffickingACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Julia G. Wittmann Rab GTP-binding proteins are involved in the regulation of distinct vesicular-transport events involving membrane targeting and fusion. They differ from other small GTPases by the presence of specific loop regions that serve as effector-binding sites in addition to the classical switch I and switch II regions. While the structures of many small GTP-binding proteins of the Ras superfamily are available in both GDP- and GTP-bound forms, Rab proteins are less well characterized than Ras proteins at the structural level. The crystallization of Rab9, a key regulatory component in the recycling of mannose-6-phosphate receptors from endosomes to the trans-Golgi network, is described here. [source] The X-ray structure of a recombinant major urinary protein at 1.75,Å resolution.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2001A comparative study of X-ray, NMR-derived structures Major urinary proteins belong to the lipocalin family and are present in the urine of rodents as an ensemble of isoforms with pheromonal activity. The crystal structure of a recombinant mouse MUP (rMUP) was solved by the molecular-replacement technique and refined to an R factor and Rfree of 20 and 26.5%, respectively, at 1.75,Å resolution. The structure was compared with an NMR model and with a crystallographic structure of the wild-type form of the protein. The crystal structures determined in different space groups present significantly smaller conformational differences amongst themselves than in comparison with NMR models. Some, but not all, of the conformational differences between the crystal and solution structures can be explained by the influence of crystallographic contacts. Most of the differences between the NMR and X-ray structures were found in the N-terminus and loop regions. A number of side chains lining the hydrophobic pocket of the molecule are more tightly packed in the NMR structure than in the crystallographic model. Surprisingly, clear and continuous electron density for a ligand was observed inside the hydrophobic pocket of this recombinant protein. Conformation of the ligand modelled inside the density is coherent with the results of recent NMR experiments. [source] Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5,Å resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sitesACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001Andrzej Wojtczak The first observation of the unique environment for thyroxine (T4) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin,thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P43212 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5,Å resolution and the structure was refined to R = 20.9% for 15,384 data in the resolution range 12,2.5,Å. Similar to human TTR, the rat protein is also a 54,000,Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains. [source] Structure of glyceraldehyde-3-phosphate dehydrogenase from the archaeal hyperthermophile Methanocaldococcus jannaschiiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Ali D. Malay The X-ray crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj-GAPDH) was determined to 1.81,Å resolution. The crystal belonged to space group C2221, with unit-cell parameters a = 83.4, b = 152.0, c = 118.6,Å. The structure was solved by molecular replacement and was refined to a final R factor of 17.1% (Rfree = 19.8%). The final structure included the cofactor NADP+ at the nucleotide-binding site and featured unoccupied inorganic and substrate phosphate-binding sites. A comparison with GAPDH structures from mesophilic sources suggested that Mj-GAPDH is stabilized by extensive electrostatic interactions between the C-terminal ,-helices and various distal loop regions, which are likely to contribute to thermal stability. The key phosphate-binding residues in the active site of Mj-GAPDH are conserved in other archaeal GAPDH proteins. These residues undergo a conformational shift in response to occupancy of the inorganic phosphate site. [source] LpxA: A natural nanotube,BIOPOLYMERS, Issue 10 2010Atanu Das Abstract UDP-N-acetylglucosamine 3-O-acyltransferase is a protein with a left-handed parallel ,-helix, which is a natural nanotube. They are associated with unusual high stability. To identify the reason behind the structural stability of ,-helical nanotubular structure, we have performed a total of 4 ,s molecular dynamics simulations of the protein in implicit solvent at four different temperatures and monitored the unfolding pathway. The correlation in movement between different regions of the nanotubular structure has been identified from the dynamical cross-correlation map and contribution of some specific residues towards unfolding transition has been identified by principal component analysis. Difference in stability of the three loop regions has also been characterized. Construction of the unfolding conformational energy landscape identifies the probable intermediates that can appear in the unfolding pathway of the protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 845,853, 2010. [source] Changes in protein conformation and dynamics upon complex formation of brain-derived neurotrophic factor and its receptor: Investigation by isotope-edited Fourier transform IR spectroscopyBIOPOLYMERS, Issue 1 2002Tiansheng Li Abstract The interactions of brain-derived neurotrophic factor (BDNF) with the extracellular domain of its receptor (trkB) are investigated by employing isotope-edited Fourier transform IR (FTIR) spectroscopy. The protein secondary structures of individual BDNF and trkB in solutions are compared with those in their complex. The temperature dependence of the secondary structures of BDNF, trkB, and their complex is also investigated. Consistent with the crystal structure, we observe by FTIR spectroscopy that BDNF in solution contains predominantly , strands (,53%) and relatively low contents of other secondary structures including , turns (,16%), disordered structures (,12%), and loops (,18%) and is deficient in , helix. We also observe that trkB in solution contains mostly , strands (52%) and little , helix. Conformational changes in both BDNF and trkB are observed upon complex formation. Specifically, upon binding of BDNF, the conformational changes in trkB appear to involve mostly , turns and disordered structures while the majority of the ,-strand conformation remains unchanged. The IR data indicate that some of the disordered structures in the loop regions are likely converted to , strands upon complex formation. The FTIR spectral data of BDNF, trkB, and their complex indicate that more amide NH groups of trkB undergo H,D exchange within the complex than those of the ligand-free receptor and that the thermal stability of trkB is decreased slightly upon binding of BDNF. The FT-Raman spectra of BDNF, trkB, and their complex show that the six intramolecular disulfide bonds of trkB undergo significant conformational changes upon binding of BDNF as a result of changes in the tertiary structure of trkB. Taken together, the FTIR and Raman data are consistent with the loosening of the tertiary structure of trkB upon binding of BDNF, which leads to more solvent exposure of the amide NH group and decreased thermal stability of trkB. This finding reveals an intriguing structural property of the neurotrophin ligand,receptor complex that is in contrast to other ligand,receptor complexes such as a cytokine,receptor complex that usually shows protection of the amide NH group and increased thermal stability upon complex formation. © 2002 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 67: 10,19, 2002; DOI 10.1002/bip.10038 [source] Structure of cyclophilin from Leishmania donovani at 1.97,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2007V. Venugopal The crystal structure of cyclophilin from Leishmania donovani (LdCyp) has been determined and refined at 1.97,Å resolution to a crystallographic R factor of 0.178 (Rfree = 0.197). The structure was solved by molecular replacement using cyclophilin from Trypanosoma cruzi as the search model. LdCyp exhibits complete structural conservation of the cyclosporin-binding site with respect to the homologous human protein, as anticipated from LdCyp,cyclosporin binding studies. Comparisons with other cyclophilins show deviations primarily in the loop regions. The solvent structure encompassing the molecule has also been analyzed in some detail. [source] | ||||||||||||||||||||||