共查询到20条相似文献,搜索用时 31 毫秒
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Wilson KA Kalkum M Ottesen J Yuzenkova J Chait BT Landick R Muir T Severinov K Darst SA 《Journal of the American Chemical Society》2003,125(41):12475-12483
Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail. 相似文献
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Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide that has an unusual threaded lasso structure in which the C-terminal "tail" of the peptide is fed through a macrocyclic "ring" formed by the N-terminal residues. Production of MccJ25 in Escherichia coli is dependent upon a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of mcjA and mcjD, thus permitting independent control of MccJ25 production and export/immunity in E. coli. We used this system to screen saturation mutagenesis libraries targeted to either the ring or tail portions of MccJ25 and discovered nearly 100 new MccJ25 variants that retain antimicrobial function. While multiple amino acid substitutions in the tail portion of the peptide are well-tolerated, mutagenesis of the ring portion of the peptide is detrimental to the antimicrobial function of MccJ25. We demonstrated that the decreased function of the ring variants is due to the inability of these variants to be transported to the cytoplasm of susceptible strains. Additionally, we found several MccJ25 variants from the tail library with improved efficacy toward the MccJ25-sensitive strains E. coli and Salmonella enterica serovar Newport with the best variants exhibiting a nearly 5-fold increase in potency. The results described here provide further evidence that diverse amino acid sequences can be tolerated by the rigid lasso peptide fold. 相似文献
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Duquesne S Destoumieux-Garzón D Zirah S Goulard C Peduzzi J Rebuffat S 《Chemistry & biology》2007,14(7):793-803
Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg(2+)-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria. 相似文献
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Zirah S Afonso C Linne U Knappe TA Marahiel MA Rebuffat S Tabet JC 《Journal of the American Society for Mass Spectrometry》2011,22(3):467-479
Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrolactam ring. The structural characterization of lasso structures and differentiation from their unthreaded
topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we
investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded
topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under
collision-induced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure.
The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry
(ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECD
spectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD
was informative and showed different extent of hydrogen migration (formation of c•/z from c/z•) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series
of radical b-type product ions ( b¢n · ) \left( {b{\prime}_n^{ \bullet }} \right) . We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z• and y/b dissociation, in
the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide
topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture. 相似文献
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Shape-persistent arylene ethynylene macrocycles have attracted much attention in supramolecular chemistry and materials science because of their unique structures and novel properties. In this Review we describe recent examples of macrocycle synthesis by cross-coupling (Sonogashira: aryl acetylene macrocycle or Glaser: aryl diacetylene macrocycle) and dynamic covalent chemistry. The primary disadvantage of the coupling methods is the kinetically determined product distribution, since a significant portion of oligomers grow beyond the length of the cyclic targets ("overshooting"). Better results have been obtained recently by a dynamic covalent approach involving reversible metathesis reactions that afford macrocycles in one step. Mechanistic studies demonstrate that macrocycle formation is thermodynamically controlled by this route. Remaining synthetic challenges include the efficient preparation of site-specifically functionalized structures and larger, more complex two- and three-dimensional molecules. 相似文献
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Microcin J25 is a ribosomally synthesised 21-residue antimicrobial peptide produced by certain strains of enterobacteria, that adopts an extraordinary 'threaded lasso' structure. To date, the biosynthesis of this peptide is little understood. Here we report the in vitro maturation of the microcin precursor peptide into active microcin J25 for the first time. Furthermore, we show that the enzymes required for the posttranslational modification of this precursor peptide are associated with the bacterial inner membrane. 相似文献
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Efficient access: the 204-residue covalent-dimer vascular endothelial growth factor with full mitogenic activity was prepared from three unprotected peptide segments by one-pot native chemical ligations. The covalent structure of the synthetic VEGF was confirmed by precise mass measurement, and the three-dimensional structure of the synthetic protein was determined by high-resolution X-ray crystallography. 相似文献
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Iwatsuki M Tomoda H Uchida R Gouda H Hirono S Omura S 《Journal of the American Chemical Society》2006,128(23):7486-7491
Two antimycobacterial agents, lariatins A and B, were isolated from the culture broth of Rhodococcus sp. K01-B0171. Their structures were elucidated by spectral analysis and advanced protein chemical methods to be unique cyclic peptides, which consist of 18 and 20 L-amino acid residues with an internal linkage between the gamma-carboxyl group of Glu8 and the alpha-amino group of Gly1. The three-dimensional structure of lariatin A deduced from NMR data by dynamical simulated annealing method indicates that the tail segment (Trp9-Pro18) passes through the ring segment (Gly1-Glu8) to form a 'lasso' structure. 相似文献
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以粗粒化的多肽链模型进行了SARS病毒包膜中E蛋白的计算机模拟,描述了该蛋白质空间构象的概貌.首先扩展了多肽链的HP模型,使之能够用于研究在水或脂环境下蛋白质折叠的行为,并且考虑了全部氨基酸残基疏水相互作用能的差异.相关格子链的MonteCarlo模拟显示了很高的计算效率.模拟再现了蛋白质的coil-globule转变,验证了蛋白质序列分布的重要性.结果表明,在水环境中,E蛋白质空间结构由紧致的疏水内核和部分向外延伸的亲水片段组成;在脂环境中,中部疏水片段会成为向外延伸的环,而当两侧紧致的亲水片段分开时,则形成桥. 相似文献
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Nucleic acid "lariats" have been of great interest to the biological community since their discovery two decades ago as splicing intermediates in the biosynthesis of messenger RNA (lariat RNA introns). We report here the first synthesis of lariat DNA and RNA via template-mediated chemical ligation of Y-shaped oligonucleotides. The method allows for the synthesis of lariat DNA of any base composition as well as the more biologically relevant lariat RNA. Typically, branched precursors and complementary linear templates ("splints") were dissolved in an equimolar ratio at a total concentration of 10(-4) M, and ligation was promoted by addition of cyanogen bromide in a pH 7.6 buffer. The template-directed cyclization was very efficient, since the amount of circularized lariat product observed in all cases was in the 40-60% range. The lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition confirmed by MALDI-TOF mass spectrometry. Thermal denaturation and circular dichroism studies of lariat:RNA and lariat:DNA duplexes were fully supportive of the isolated "lasso" structures. Further characterization was conducted by enzymatic degradation with spleen phosphodiesterase (a 3'-exonuclease) and the RNA lariat debranching enzyme, a specific 2',5'-phosphodiesterase. 相似文献
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Three new 2-picolyloxy lariat ether compounds are prepared and solid-state structures are determined for two of them. Complexation behavior for a series of lariat ethers with pyridyl-containing side arms toward alkali metal cations and Ag+ is assessed by two-phase metal picrate extraction from aqueous solutions into chloroform and in homogeneous solutions by isothermal titration calorimetry in methanol. 相似文献
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We have designed a 21-residue α-peptide that simultaneously recognizes two decadeoxyoligothymidine (dT(10)) tracts to form triplexes with a peptide-DNA strand ratio of 1:2. The synthetic peptide side chain displays 10 melamine rings, which provide a bifacial thymine-recognition interface along the length of the 21-residue peptide. Recognition is selective for thymine over other nucleobases and drives the formation of ternary peptide·[dT(10)](2) complexes as well as heterodimeric peptide·[dT(10)C(10)T(10)] hairpin structures with triplex stems. 相似文献
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Jonsson AP Bergman T Jörnvall H Griffiths WJ Bratt P Strömberg N 《Journal of the American Society for Mass Spectrometry》2001,12(3):337-342
Tryptic digestion of the 150-residue human acidic salivary proline-rich protein 1 (PRP-1) generated eight peptides, two of which corresponded to the N-terminal 30-residue segment. In each of the other six tryptic peptides, a consensus repeat with the structure PQGPPQQGG was present. A facile Gln-Gly cleavage between the second and the third residues of the repeat was observed during collision-induced dissociation experiments. We postulate possible mechanisms to account for this reactivity, involving attack on the peptidyl carbonyl group by the Gln sidechain. Significantly, the Gln-Gly cleavage has been shown to be biologically important in the bacterial degradation of PRPs in saliva, generating bacteria-binding Pro-Gln C-termini. We suggest a link between the gas-phase chemistry and the biochemical degradation of these molecules. 相似文献
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A newchola lariat ether (1, a 21-crown-6) was constructed fromreadily available precursors. The association constant of compound1 with alkali metal picrates was measured using Cram’s extraction protocol. Evidence is presented for the involvement of the
3-methoxy group for the complexation. Energy minimised structures show that the A-ring gets slightly distorted upon metal
ion binding.
Dedicated to Professor C N R Rao on his 70th birthday 相似文献
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The solid-phase synthesis of a cyclic peptide containing the 21-residue epitope found in the A-B loop of the Cepsilon3 domain of human immunoglobulin E has been carried out. The key macrocyclization step to form the 65-membered ring is achieved in approximately 15% yield via an "on-resin" Sonogashira coupling reaction which concomitantly installs a diphenylacetylene amino acid conformational constraint within the loop. 相似文献
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This paper examines the self-assembly of cyclic β-tripeptides using density functional theory. On the basis of literature precedents, these cyclic peptides were expected to self-assemble into cylindrical structures by stacking through backbone-backbone hydrogen bonding. Our calculations show that such stacking is energetically favorable, that the association energy per cyclic peptide decreases (becomes more favorable), and that the overall macrodipole moment of the cylindrical assembly increases with the number of stacked rings, for up to eight rings. For a structure in which two peptide ring units are joined through a single side chain-side chain covalent linker, the association energy between the two rings is favorable, albeit less so than for the unlinked rings. Significantly, the association energy in the dimers is only weakly dependent on the length (above a certain minimum) and conformation of the covalent linkers. Finally, as a plausible route for controlling assembly/disassembly of nanocylinders, we show that, for a pair of rings, each bearing a single amino-functionalized side chain, protonation of the amino group results in a strongly positive (unfavorable) association energy between the two rings. 相似文献
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Current studies of lariat RNA structure and function are hindered by the lack of access to synthetic lariats. A novel approach to the synthesis of both DNA and RNA lariats is presented here. Noteworthy features of the methodology are the regiospecific formation of the 2'-5'-phosphodiester linkage, the unusual parallel stranded DNA/RNA hybrid (or parallel RNA/RNA duplex) that forms between an RNA template and a folded 22-nt DNA (or RNA) substrate, and the efficiency of the chemical ligation step at an adenosine branchpoint (50-80%). The DNA and RNA lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition were confirmed by MALDI-TOF mass spectrometry. Thermal denaturation as well as enzymatic and chemical hydrolysis fully supported the proposed lariat structures. Characterization of control parallel duplexes was conducted by gel shift assays and enzymatic degradation with RNase H. The successful synthesis of the lariat molecules described here will allow structural and biochemical studies aimed at better understanding the splicing and debranching mechanisms in which these unusual nucleic acids are involved. 相似文献
20.
Romina Oliva Abdul Rajjak Shaikh Andrea Petta Anna Vangone Luigi Cavallo 《Molecules (Basel, Switzerland)》2021,26(9)
The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The “fusion core” of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one. 相似文献