共查询到20条相似文献,搜索用时 140 毫秒
1.
Keigo Hisamatsu Tomonao Nagao Hideaki UnnoShuichiro Goda Tomomitsu Hatakeyama 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca2 +-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores.Methods
Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined.Results
K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane.Conclusions
Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III.General significance
The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins. 相似文献2.
Li Xing Meijuan Niu Xia ZhaoLawrence Kleiman 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.Methods
The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.Results
Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNALys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.Conclusions
The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.General significance
The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro. 相似文献3.
Julia Knöckel Rositsa Jordanova Ingrid B. Müller Carsten Wrenger Matthew R. Groves 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.Methods
Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.Results
Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.Conclusions
As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.General significance
The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors. 相似文献4.
Hazuki E. Miwa Thomas A. Gerken Tru D. Huynh Lori R. Duesler Meghan Cotter Thomas M. Hering 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.Methods
To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.Results
Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.Conclusion
We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.General significance
Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis. 相似文献5.
Analyses of non-leucine-rich repeat (non-LRR) regions intervening between LRRs in proteins 总被引:1,自引:0,他引:1
Norio Matsushima Tomoko Mikami Takanori Tanaka Hiroki Miyashita Keiko Yamada Yoshio Kuroki 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Many proteins have LRR (leucine-rich repeat) units interrupted by non-LRRs which we call IR (non-LRR island region).Methods
We identified proteins containing LRR@IRs (LRRs having IR) by using a new method and then analyzed their natures and distributions.Results
LRR@IR proteins were found in over two hundred proteins from prokaryotes and from eukaryotes. These are divided into twenty-one different protein families. The IRs occur one to four times in LRR regions and range in length from 5 to 11,265 residues. The IR lengths in Fungi adenylate cyclases (acys) range from 5 to 116 residues; there are 22 LRR repeats. The IRs in Leishmania proteophosphoglycans (ppgs) vary from 105 to 11,265 residues. These results indicate that the IRs evolved rapidly. A group of LRR@IR proteins—LRRC17, chondroadherin-like protein, ppgs, and four Pseudomonas proteins—have a super motif consisting of an LRR block and its adjacent LRR@IR region. This indicates that the entire super motif experienced duplication. The sequence analysis of IRs offers functional similarity in some LRR@IR protein families.General significance
This study suggests that various IRs and super motifs provide a great variety of structures and functions for LRRs. 相似文献6.
Demei Meng Lin Shen Rui Yang Xinhua Zhang Jiping Sheng 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
1-Aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme that catalyses the final step in the biosynthesis of the plant hormone ethylene. Recently, the first ACO homologue gene was isolated in Agaricus bisporus, whereas information concerning the nature of the ethylene-forming activity of this mushroom ACO is currently lacking.Methods
Recombinant ACO from A. bisporus (Ab-ACO) was purified and characterised for the first time. Molecular modelling combined with site-directed mutagenesis and kinetic and spectral analysis were used to investigate the property of Ab-ACO.Results
Ab-ACO has eight amino acid residues that are conserved in the Fe (II) ascorbate family of dioxygenases, including four catalytic residues in the active site, but Ab-ACO lacks a key residue, S289. In comparison to plant ACOs, Ab-ACO requires ACC and Fe (II) but does not require ascorbate. In addition, Ab-ACO had relatively low activity and was completely dependent on bicarbonate, which could be ascribed to the replacement of S289 by G289. Moreover, the ferrous ion could induce a change in the tertiary, but not the secondary, structure of Ab-ACO.Conclusions
These results provide crucial experimental support for the ability of Ab-ACO to catalyse ethylene formation in a similar manner to that of plant ACOs, but there are differences between the biochemical and catalytic characteristics of Ab-ACO and plant ACOs.General significance
This work enhances the understanding of the ethylene biosynthesis pathways in fungi and could promote profound physiological research of the role of ethylene in the regulation of mushroom growth and development. 相似文献7.
David Delvaux Frédéric Kerff Mamidanna R.V.S. Murty Bernard Lakaye Jan Czerniecki Gregory Kohn Pierre Wins Raphaël Herman Valérie Gabelica Fabien Heuze Xavier Tordoir Raphaël Marée André Matagne Paulette Charlier Edwin De Pauw Lucien Bettendorff 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates.Methods
Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase.Results
Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine–tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds.Conclusions
The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals.General significance
We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins. 相似文献8.
Background
Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.Methods
Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.Results
A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).Conclusion
With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen. 相似文献9.
Yoshimitsu Kakuta Kazuhiro Usuda Takashi Nakashima Makoto Kimura Yoichi Aso Kohji Yamamoto 《Biochimica et Biophysica Acta (BBA)/General Subjects》2011
Background
Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues.Methods
The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu.Results
We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features.Conclusions
This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function.General Significance
Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance. 相似文献10.
Xiaobin Huang Leslie R. Morse Yan Xu Jaromir Zahradka Hana Sychrová Phil Stashenko Feiyue Fan Ricardo A. Battaglino 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes.Methods
The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth.Results
Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4 M NaCl, suggesting that these residues are also essential for antiporter activity.Conclusions
Evolutionarily conserved amino acids are required for full antiporter function.General Significance
Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo. 相似文献11.
Nicola Giangregorio Ferdinando Palmieri Cesare Indiveri 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
The mitochondrial carnitine/acylcarnitine carrier (CAC) is essential for cell metabolism since it catalyzes the transport of acylcarnitines into mitochondria allowing the β-oxidation of fatty acids. CAC functional and structural properties have been characterized. Cys residues which could form disulfides suggest the involvement of CAC in redox switches.Methods
The effect of GSH and GSSG on the [3H]-carnitine/carnitine antiport catalyzed by the CAC in proteoliposomes has been studied. The Cys residues involved in the redox switch have been identified by site-directed mutagenesis. Glutathionylated CAC has been assessed by glutathionyl-protein specific antibody.Results
GSH led to increase of transport activity of the CAC extracted from liver mitochondria. A similar effect was observed on the recombinant CAC. The presence of glutaredoxin-1 (Grx1) accelerated the GSH activation of the recombinant CAC. The effect was more evident at 37 °C. GSSG led to transport inhibition which was reversed by dithioerythritol (DTE). The effects of GSH and GSSG were studied on CAC Cys-mutants. CAC lacking C136 and C155 was insensitive to both reagents. Mutants containing these two Cys responded as the wild-type. Anti-glutathionyl antibody revealed the formation of glutathionylated CAC.Conclusions
CAC is redox-sensitive and it is regulated by the GSH/GSSG couple. C136 and C155 are responsible for the regulation which occurs through glutathionylation.General significance
CAC is sensitive to the redox state of the cell switching between oxidized and reduced forms in response to variation of GSSG and GSH concentrations. 相似文献12.
13.
Geumsoo Kim Stephen J. Weiss Rodney L. Levine 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Cysteine and methionine are the two sulfur containing amino acids in proteins. While the roles of protein-bound cysteinyl residues as endogenous antioxidants are well appreciated, those of methionine remain largely unexplored.Scope
We summarize the key roles of methionine residues in proteins.Major conclusion
Recent studies establish that cysteine and methionine have remarkably similar functions.General significance
Both cysteine and methionine serve as important cellular antioxidants, stabilize the structure of proteins, and can act as regulatory switches through reversible oxidation and reduction. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. 相似文献14.
Tricia A. Ulmer Vicki Keeler Sabine André Hans-Joachim Gabius Lambert Loh Suzanne Laferté 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9.Methods
The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis.Results
TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium.Conclusion
TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells.General significance
Proteolytic cleavage of TAA90K may have functional implications in colon cancer. 相似文献15.
16.
Ernesto Ortiz Martha Rendón-Anaya Solange Cristina Rego Elisabeth Ferroni Schwartz Lourival Domingos Possani 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
The venoms of several scorpion species have long been associated with pancreatitis in animal models and humans. Antarease, a Zn-metalloprotease from Tityus serrulatus, is able to penetrate intact pancreatic tissue and disrupts the normal vesicular traffic necessary for secretion, so it could play a relevant role in the onset of acute pancreatitis.Methods
The cDNA libraries from five different scorpion species were screened for antarease homologs with specific primers. The amplified PCR products were cloned and sequenced. A structural model was constructed to assess the functionality of the putative metalloproteases. A phylogenetic analysis was performed to identify clustering patterns of these venom components.Results
Antarease-like sequences were amplified from all the screened cDNA libraries. The complete sequence of the antarease from T. serrulatus was obtained. The structural model of the putative antarease from Tityus trivittatus shows that it may adopt a catalytically active conformation, sharing relevant structural elements with previously reported metalloproteases of the ADAM family. The phylogenetic analysis reveals that the reported sequences cluster in groups that correlate with the geographical localization of the respective species.Conclusions
Antareases are ubiquitous to a broad range of scorpion species, where they could be catalytically active enzymes. These molecules can be used to describe the evolution of scorpion venoms under different ecogeographic constrains.General significance
For the first time the complete sequence of the antareases is reported. It is demonstrated that antareases are common in the venom of different scorpion species. They are now proposed as targets for antivenom therapies. 相似文献17.
Inka Brockhausen Thomas Dowler Hans Paulsen 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.Methods
We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.Results
Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.Conclusions
The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.General significance
Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease. 相似文献18.
Lv Z Zhang X Liu L Chen J Nie Z Sheng Q Zhang W Jiang C Yu W Wang D Wu X Zhang S Li J Zhang Y 《Gene》2012,502(2):118-124
Background
Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown.Methods
To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae. An His-tagged BmPHB fusion protein was expressed in Escherichia coli Rosetta (DE3) and purified with affinity and reversed-phase chromatography. Purified rBmPHB was used to generate anti-BmPHB polyclonal antibody. The subcellular localization of BmPHB was analysed by immunohistochemistry.Results
BmPHB gene has an ORF of 825 bp, encoding a predicted peptide with 274 amino acid residues. Immunostaining indicate that prohibitin is expressed in nucleus and predominately in cytoplasm. Western blot analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine, and head. However, no expression was detected in larva's silk gland and epidermis. In addition, BmPHB was expressed in the nascent egg, larva and pupa, but not in the moth.Conclusions
The expression of BmPHB gene presents differential characteristic in different stage and tissues. It may play important roles in the development of silkworm.General significance
Studies on prohibitin have been still restricted to a few specific insects and insect cell lines such as Drosophila, Acyrthosiphon pisum and mosquito cell lines, not yet in silkworm. This is a first characterization of prohibitin in silkworm, B. mori. 相似文献19.
Eva Macakova Miroslava Kopecka Zdenek Kukacka Dana Veisova Petr Novak Petr Man Tomas Obsil Veronika Obsilova 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Trehalases are highly conserved enzymes catalyzing the hydrolysis of trehalose in a wide range of organisms. The activity of yeast neutral trehalase Nth1 is regulated in a 14-3-3- and a calcium-dependent manner. The Bmh proteins (the yeast 14-3-3 isoforms) recognize phosphorylated Nth1 and enhance its enzymatic activity through an unknown mechanism.Methods
To investigate the structural basis of interaction between Nth1 and Bmh1, we used hydrogen/deuterium exchange coupled to mass spectrometry, circular dichroism spectroscopy and homology modeling to identify structural changes occurring upon the complex formation.Results
Our results show that the Bmh1 protein binding affects structural properties of several regions of phosphorylated Nth1: the N-terminal segment containing phosphorylation sites responsible for Nth1 binding to Bmh, the region containing the calcium binding domain, and segments surrounding the active site of the catalytic trehalase domain. The complex formation between Bmh1 and phosphorylated Nth1, however, is not accompanied by the change in the secondary structure composition but rather the change in the tertiary structure.Conclusions
The 14-3-3 protein-dependent activation of Nth1 is based on the structural change of both the calcium binding domain and the catalytic trehalase domain. These changes likely increase the accessibility of the active site, thus resulting in Nth1 activation.General significance
The results presented here provide a structural view of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1, which might be relevant to understand the process of Nth1 activity regulation as well as the role of the 14-3-3 proteins in the regulation of other enzymes. 相似文献20.
Iveta Uhliariková Mária Vršanská Barry V. McCleary Peter Biely 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013