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1.
To ensure fidelity of translation, several aminoacyl-tRNA synthetases (aaRSs) possess editing capability to hydrolyse mis-aminoacylated tRNAs. In this report, based on our previously-modelled structure of leucyl-tRNA synthetase (LeuRS) complexed with valyl-tRNALeu, further structural modelling has been performed along with molecular dynamics simulations. This enabled the identification of the nucleophile, which is different from that suggested by the crystal structure of the LeuRS • Nva2AA complex. Our results revealed that the 3′ hydroxyl group of A76 acts as a “gate” to regulate the accessibility of the nucleophile; thus, the opening of the gate leads to the productive complex for the reaction. 相似文献
2.
Xiao-Long Zhou 《Biochemical and biophysical research communications》2009,386(3):510-333
Leucyl-tRNA synthetase (LeuRS) is responsible for the Leu-tRNALeu synthesis. The connective peptide 1 (CP1) domain inserted into the Rossmann nucleotide binding fold possesses editing active site to hydrolyze the mischarged tRNALeu with noncognate amino acid, then to ensure high fidelity of protein synthesis. A few co-crystal structures of LeuRS with tRNALeu in different conformations revealed that tRNALeu 3′ end shuttled between synthetic and editing active sites dynamically with direct and specific interaction with the CP1 domain. Here, we reported that Y515 and Y520 outside the editing active site of CP1 domain of Giardia lamblia LeuRS (GlLeuRS) are crucial for post-transfer editing by influencing the binding affinity with mischarged tRNALeu. Mutations on Y515 and Y520 also decreased tRNALeu charging activity to various extents but had no effect on leucine activation. Our results gave some biochemical knowledge about interaction of tRNALeu 3′ end with the CP1 domain in archaeal/eukaryotic LeuRS. 相似文献
3.
Anton Iershov Konstantin Odynets Alexander Kornelyuk Vadim Kavsan 《Central European Journal of Biology》2010,5(4):407-420
The human genome encodes six proteins of family 18 glycosyl hydrolases, two active chitinases and four chitinase-like lectins
(chi-lectins) lacking catalytic activity. The present article is dedicated to homology modeling of 3D structure of human chitinase
3-like 2 protein (CHI3L2), which is overexpressed in glial brain tumors, and its structural comparison with homologous chi-lectin
CHI3L1. Two crystal structures of CHI3L1 in free state (Protein Data Bank codes 1HJX and 1NWR) were used as structural templates
for the homology modeling by Modeller 9.7 program, and the best quality model structure was selected from the obtained model
ensemble. Analysis of potential oligosaccharide-binding groove structures of CHI3L1 and CHI3L2 revealed significant differences
between these two homologous proteins. 8 of 19 amino acid residues important for ligand binding are substituted in CHI3L2:
Tyr34/Asp39, Trp69/Lys74, Trp71/Lys76, Trp99/Tyr104, Asn100/Leu105, Met204/Leu210, Tyr206/Phe212 and Arg263/His271. The differences between these residues could influence the structure of the ligand-binding groove and substantially change
the ability of CHI3L2 to bind oligosaccharide ligands. 相似文献
4.
Xiangfeng Meng Justyna M. Dobruchowska Tjaard Pijning Cesar A. López Johannis P. Kamerling Lubbert Dijkhuizen 《The Journal of biological chemistry》2014,289(47):32773-32782
Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu940 in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu940 of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67–100% of (α1→6) linkages) were produced by GTF180 and its Leu940 mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu940 in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes. 相似文献
5.
A unique C-terminal domain extension is required by most leucyl-tRNA synthetases (LeuRS) for aminoacylation. In one exception, the enzymatic activity of yeast mitochondrial LeuRS is actually impeded by its own C-terminal domain. It was proposed that the yeast mitochondrial LeuRS has compromised its aminoacylation activity to some extent and adapted its C terminus for a second role in RNA splicing, which is also essential. X-ray crystal structures of the LeuRS-tRNA complex show that the 60 residue C-terminal domain is tethered to the main body of the enzyme via a flexible peptide linker and allows interactions with the tRNALeu elbow. We hypothesized that this short peptide linker would facilitate rigid body movement of the C-terminal domain as LeuRS transitions between an aminoacylation and editing complex or, in the case of yeast mitochondrial LeuRS, an RNA splicing complex. The roles of the C-terminal linker peptide for Escherichia coli and yeast mitochondrial LeuRS were investigated via deletion mutagenesis as well as by introducing chimeric swaps. Deletions within the C-terminal linker of E. coli LeuRS determined that its length, rather than its sequence, was critical to aminoacylation and editing activities. Although deletions in the yeast mitochondrial LeuRS peptide linker destabilized the protein in general, more stable chimeric enzymes that contained an E. coli LeuRS C-terminal domain showed that shortening its tether stimulated aminoacylation activity. This suggested that limiting C-terminal domain accessibility to tRNALeu facilitates its role in protein synthesis and may be a unique adaptation of yeast mitochondrial LeuRS that accommodates its second function in RNA splicing. 相似文献
6.
Elena Seiradake Weimin Mao Stephen J. Baker M.R.K. Alley 《Journal of molecular biology》2009,390(2):196-1250
Leucyl-tRNA synthetase (LeuRS) specifically links leucine to the 3′ end of tRNAleu isoacceptors. The overall accuracy of the two-step aminoacylation reaction is enhanced by an editing domain that hydrolyzes mischarged tRNAs, notably ile-tRNAleu. We present crystal structures of the editing domain from two eukaryotic cytosolic LeuRS: human and fungal pathogen Candida albicans. In comparison with previous structures of the editing domain from bacterial and archeal kingdoms, these structures show that the LeuRS editing domain has a conserved structural core containing the active site for hydrolysis, with distinct bacterial, archeal, or eukaryotic specific peripheral insertions. It was recently shown that the benzoxaborole antifungal compound AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-1,2-benzoxaborole) inhibits LeuRS by forming a covalent adduct with the 3′ adenosine of tRNAleu at the editing site, thus locking the enzyme in an inactive conformation. To provide a structural basis for enhancing the specificity of these benzoxaborole antifungals, we determined the structure at 2.2 Å resolution of the C. albicans editing domain in complex with a related compound, AN3018 (6-(ethylamino)-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol), using AMP as a surrogate for the 3′ adenosine of tRNAleu. The interactions between the AN3018-AMP adduct and C. albicans LeuRS are similar to those previously observed for bacterial LeuRS with the AN2690 adduct, with an additional hydrogen bond to the extra ethylamine group. However, compared to bacteria, eukaryotic cytosolic LeuRS editing domains contain an extra helix that closes over the active site, largely burying the adduct and providing additional direct and water-mediated contacts. Small differences between the human domain and the fungal domain could be exploited to enhance fungal specificity. 相似文献
7.
Morana Dulic Nevena Cvetesic Igor Zivkovic Andrés Palencia Stephen Cusack Branimir Bertosa Ita Gruic-Sovulj 《Journal of molecular biology》2018,430(1):1-16
The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNALeu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNALeu differed by about 104-fold. We further established the critical role for the A76 3′-OH group of the tRNALeu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNALeu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection. 相似文献
8.
Montasir Elahi Monirul M. Islam Keiichi Noguchi Masafumi Yohda Hiroyuki Toh Yutaka Kuroda 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(3):585-592
Dengue viruses (DEN) are classified into four serotypes (DEN1-DEN4) exhibiting high sequence and structural similarities, and infections by multiple serotypes can lead to the deadly dengue hemorrhagic fever. Here, we aim at characterizing the thermodynamic stability of DEN envelope protein domain III (ED3) during its evolution, and we report a structural analysis of DEN4wt ED3 combined with a systematic mutational analysis of residues 310 and 387. Molecular modeling based on our DEN3 and DEN4 ED3 structures indicated that the side-chains of residues 310/387, which are Val310/Ile387 and Met310/Leu387 in DEN3wt and DEN4wt, respectively, could be structurally compensated, and that a “size switch type repacking” might have occurred at these sites during the evolution of DEN into its four serotypes. This was experimentally confirmed by a 10 °C and 5 °C decrease in the thermal stability of, respectively, DEN3 ED3 variants with Met310/Ile387 and Val310/Leu387, whereas the variant with Met310/Leu387, which contains a double mutation, had the same stability as the wild type DEN3. Namely, the Met310Val mutation should have preceded the Leu387Ile mutation in order to maintain the tight internal packing of ED3 and thus its thermodynamic stability. This view was confirmed by a phylogenetic reconstruction indicating that a common DEN ancestor would have Met310/Leu387, and the intermediate node protein, Val310/Leu387, which then mutated to the Val310/Ile387 pair found in the present DEN3. The hypothesis was further confirmed by the observation that all of the present DEN viruses exhibit only stabilizing amino acid pairs at the 310/387 sites. 相似文献
9.
Anusha P. Subasinghage J. Michael Conlon Chandralal M. Hewage 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):1020-1028
Peptide XT-7 (GLLGP5LLKIA10AKVGS15NLL.NH2) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC50 = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC50 > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d3)-H2O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu3 and Leu17 whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu6 and Leu17. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu3 and Leu17. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H2O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly4 → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7. 相似文献
10.
Laurent Malivert Virginie Ropars Marcela Nunez Pascal Drevet Simona Miron Guilhem Faure Raphael Guerois Jean-Paul Mornon Patrick Revy Jean-Baptiste Charbonnier Isabelle Callebaut Jean-Pierre de Villartay 《The Journal of biological chemistry》2010,285(34):26475-26483
In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor. Ten of 27 screened mutants were nonfunctional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg64, Leu65, and Leu115) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4. 相似文献
11.
Chandralal M. Hewage Lu Jiang John A. Parkinson Robert Ramage Ian H. Sadler 《Journal of biomolecular structure & dynamics》2013,31(2):425-435
Abstract The solution structure of a synthetic ETB selective agonist, ET-l[Cys(Acm)1,15, Ala3, Leu7, dAsp8, Aib11] has been solved by 1H NMR and molecular modelling studies. Such solution structures of linear modified peptides in aqueous methanol are being used in an ongoing program of research designed to assist in an understanding of the basic structural requirements for the biological activity of vasoconstrictors. The resulting structure of this peptide is characterised by an α-helical conformation between residues Leu6-His16 and by N- and C-termi- ni which assume no defined conformation. A knowledge of the solution structures of this and related peptides, which are ETB selective agonists, are proving to be important in the understanding of how they interact with the ETB receptor. 相似文献
12.
The millisecond conformational flexibility is functionally important for nucleic acids and can be studied through probing the base pair open-close kinetics by proton exchange nuclear magnetic resonance (NMR) spectroscopy. Here, the traditional imino proton exchange NMR experiments were modified with transverse relaxation optimized spectroscopy and were applied to accurately measure imino proton exchange rates of all base pairs in Escherichia coli tRNALeu (CAG), and their dependence on magnesium ion concentration. Finally, we correlated millisecond conformational flexibility with aminoacylation of tRNALeu and proposed that the flexibility of the acceptor stem and the core region might contribute to aminoacylation of tRNALeu. 相似文献
13.
Hodek P Karabec M Sulc M Sopko B Smrcek S Martínek V Hudecek J Stiborová M 《Archives of biochemistry and biophysics》2007,468(1):82-91
To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu359-Lys373), peptide II (Leu30-Arg48), peptide III (Phe127-Arg140), and peptide IV (Arg434-Arg443). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg133, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding. 相似文献
14.
Qian Huang Xiao-Long Zhou Qin-Hua Hu Hui-Yan Lei Zhi-Peng Fang Peng Yao En-Duo Wang 《RNA (New York, N.Y.)》2014,20(9):1440-1450
Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNALeu. LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain–domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS. 相似文献
15.
Changes in isoaccepting species of tRNALeu were determined in germinating pea seedlings and in developing pods. Leucine specific transfer ribonucleic acids of pea cotyledons can be fractionated into four isoaccepting species by reversed-phase chromatography (RPC-5) on a Plaskon column. In contrast, only two species of tRNALeu were observed in developing seed pods. Leucyl-tRNA synthetase purified by ammonium sulfate precipitation and DEAE cellulose column chromatography retained the full range of specificity towards all four tRNALeu species of pea cotyledons. This partially purified pea cotyledon enzyme could be further separated on a hydroxylapatite (HA) column into two peaks of leucyl-tRNA synthetase activity. Enzyme 1 is dominant in seed pods while 2 is predominant in cotyledons. Enzymes 1 and 2 from cotyledons were examined for the amino acid acceptor activity of twelve different amino acids. Both these fractions showed less than 3% acceptor activity for eleven other amino acids as compared to leucine-tRNA synthetase activity. Preliminary characterization of enzyme 2 from cotyledon, by isoelectric focusing and polyacrylamide gel electrophoresis indicates at least three subspecies. 相似文献
16.
I. A. Kostanyan S. S. Zhokhov M. V. Astapova S. M. Dranitsyna A. P. Bogachuk L. K. Baidakova I. L. Rodionov I. I. Baskin O. N. Golubeva J. Tombran-Tink V. M. Lipkin 《Russian Journal of Bioorganic Chemistry》2000,26(9):505-511
It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the
human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in itsC-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41–46
fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and
synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm ofXenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides
and the computer models of the spatial structures of the full-size PEDF and the PEDF with theC-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward. 相似文献
17.
Afaf Mikou Steven R. LaPlante Eric Guittet Jean-Yves Lallemand Marie-France Martin-Eau Claire Hervé Rochat 《Journal of biomolecular NMR》1992,2(1):57-70
Summary
1H NMR has been applied to a3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpionAndroctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20–28) which is linked by two disulfide bridges to the central strand of a triple-stranded antiparallel -sheet. Turns were identified at residues 15–17, 47–49 and also at residues 51–53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu37, Leu23, Val47, Tyr14, Trp45 and Tyr5. The Trp45 and Tyr5 rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.Thenomenclature used is similar to that described by Wütrich, 1986. 相似文献
18.
Nathan R. West Benjamin Farnell Jill I. Murray Fraser Hof Peter H. Watson Martin J. Boulanger 《Protein science : a publication of the Protein Society》2009,18(12):2615-2623
S100A7 (psoriasin) is a calcium‐ and zinc‐binding protein implicated in breast cancer. We have shown previously that S100A7 enhances survival mechanisms in breast cells through an interaction with c‐jun activation domain binding protein 1 (Jab1), and an engineered S100A7 triple mutant (Asp56Gly, Leu78Met, and Gln88Lys—S100A73) ablates Jab1 binding. We extend these results to include defined breast cancer cell lines and demonstrate a disrupted S100A73/Jab1 phenotype is maintained. To establish the basis for the abrogated Jab1 binding, we have recombinantly produced S100A73, demonstrated that it retains the ability to form an exceptionally thermostable dimer, and solved the three dimensional crystal structure to 1.6 Å. Despite being positioned at the dimer interface, the Leu78Met mutation is easily accommodated and contributes to a methionine‐rich pocket formed by Met12, Met15, and Met34. In addition to altering the surface charge, the Gln88Lys mutation results in a nearby rotameric shift in Tyr85, leading to a substantially reorganized surface cavity and may influence zinc binding. The final mutation of Asp56 to Gly results in the largest structural perturbation shortening helix IV by one full turn. It is noteworthy that position 56 lies in one of two divergent clusters between S100A7 and the functionally distinct yet highly homologous S100A15. The structure of S100A73 provides a unique perspective from which to characterize the S100A7‐Jab1 interaction and better understand the distinct functions between S100A7, and it is closely related paralog S100A15. 相似文献
19.
We have constructed a model of the complex between tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and tRNATyr by successive cycles of predictions, mutagenesis of TyrRS and molecular modeling. We confront this model with data obtained independently, compare it to the crystal structures of other complexes and review recent data on the discrimination between tRNAs by TyrRS. Comparison of the crystal structures of TyrRs and GlnRS, both of which are class I synthetases, and comparison of the identity elements of tRNATyr and tRNAGln indicate that the two synthetases bind their cognate tRNAs differently. The mutagenesis data on tRNATyr confirm the model of the TyrRS:tRNATyr complex on the following points. TyrRS approaches tRNATyr on the side of the variable loop. The bases of the first three pairs of the acceptor stem are not recognized. The presence of the NH2 group in position C6 and the absence of a bulky group in position C2 are important for the recognition of the discriminator base A73 by TyrRS, which is fully realized only in the transition state for the acyl transfer. The anticodon is the major identity element of tRNATyr. We have set up an in vivo approach to study the effects of synthetase mutations on the discrimination between tRNAs. Using this approach, we have shown that residue Glul52 of TyrRS acts as a purely negative discriminant towards non-cognate tRNAs, by electrostatic and steric repulsions. The overproductions of the wild type TyrRSs from E coli and B stearothermophilus are toxic to E coli, due to the mischarging or the non-productive binding of tRNAs. The construction of a family of hybrids between the TyrRSs from E coli and B stearothermophilus has shown that their sequences and structures have remained locally compatible through evolution, for holding and function, in particular for the specific recognition and charging of tRNATyr. 相似文献
20.
Kim E Youn B Kemper L Campbell C Milting H Varsanyi M Kang C 《Journal of molecular biology》2007,373(4):1047-1057
Mutations of conserved residues of human cardiac calsequestrin (hCSQ2), a high-capacity, low-affinity Ca2+-binding protein in the sarcoplasmic reticulum, have been associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). In order to understand the molecular mechanism and pathophysiological link between these CPVT-related missense mutations of hCSQ2 and the resulting arrhythmias, we generated three CPVT-causing mutants of hCSQ2 (R33Q, L167H, and D307H) and two non-pathological mutants (T66A and V76M) and investigated the effect of these mutations. In addition, we determined the crystal structure of the corresponding wild-type hCSQ2 to gain insight into the structural effects of those mutations. Our data show clearly that all three CPVT-related mutations lead to significant reduction in Ca2+-binding capacity in spite of the similarity of their secondary structures to that of the wild-type hCSQ2. Light-scattering experiments indicate that the Ca2+-dependent monomer-polymer transitions of the mutants are quite different, confirming that the linear polymerization behavior of CSQ is linked directly to its high-capacity Ca2+ binding. R33Q and D307H mutations result in a monomer that appears to be unable to form a properly oriented dimer. On the other hand, the L167H mutant has a disrupted hydrophobic core in domain II, resulting in high molecular aggregates, which cannot respond to Ca2+. Although one of the non-pathological mutants, T66A, shares characteristics with the wild-type, the other null mutant, V76M, shows significantly altered Ca2+-binding and polymerization behaviors, calling for careful reconsideration of its status. 相似文献