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1.  FILTREST3D: discrimination of structural models using restraints from experimental data 
Bioinformatics  2010;26(23):2986-2987.
Summary: Automatic methods for macromolecular structure prediction (fold recognition, de novo folding and docking programs) produce large sets of alternative models. These large model sets often include many native-like structures, which are often scored as false positives. Such native-like models can be more easily identified based on data from experimental analyses used as structural restraints (e.g. identification of nearby residues by cross-linking, chemical modification, site-directed mutagenesis, deuterium exchange coupled with mass spectrometry, etc.). We present a simple server for scoring and ranking of models according to their agreement with user-defined restraints.
Availability: FILTREST3D is freely available for users as a web server and standalone software at: http://filtrest3d.genesilico.pl/
Contact: iamb@genesilico.pl
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq582
PMCID: PMC2982159  PMID: 20956242
2.  Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair 
Human mutation  2010;31(8):975-982.
Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome which predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C-terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1-PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1-CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased co-expression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations.
doi:10.1002/humu.21301
PMCID: PMC2908215  PMID: 20533529
Lynch syndrome; HNPCC; MLH1; PMS2; MutL; missense mutation; dimerization
3.  Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit 
Nucleic Acids Research  2010;39(5):1903-1918.
NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.
doi:10.1093/nar/gkq1033
PMCID: PMC3061052  PMID: 21062819
4.  REPAIRtoire—a database of DNA repair pathways 
Nucleic Acids Research  2010;39(Database issue):D788-D792.
REPAIRtoire is the first comprehensive database resource for systems biology of DNA damage and repair. The database collects and organizes the following types of information: (i) DNA damage linked to environmental mutagenic and cytotoxic agents, (ii) pathways comprising individual processes and enzymatic reactions involved in the removal of damage, (iii) proteins participating in DNA repair and (iv) diseases correlated with mutations in genes encoding DNA repair proteins. REPAIRtoire provides also links to publications and external databases. REPAIRtoire contains information about eight main DNA damage checkpoint, repair and tolerance pathways: DNA damage signaling, direct reversal repair, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination repair, nonhomologous end-joining and translesion synthesis. The pathway/protein dataset is currently limited to three model organisms: Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. The DNA repair and tolerance pathways are represented as graphs and in tabular form with descriptions of each repair step and corresponding proteins, and individual entries are cross-referenced to supporting literature and primary databases. REPAIRtoire can be queried by the name of pathway, protein, enzymatic complex, damage and disease. In addition, a tool for drawing custom DNA–protein complexes is available online. REPAIRtoire is freely available and can be accessed at http://repairtoire.genesilico.pl/.
doi:10.1093/nar/gkq1087
PMCID: PMC3013684  PMID: 21051355
5.  New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA 
Nucleic Acids Research  2010;38(19):6533-6543.
Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N1-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N1 atom of a purine residue is discussed.
doi:10.1093/nar/gkq451
PMCID: PMC2965216  PMID: 20525789
6.  Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases 
Nucleic Acids Research  2010;38(12):4120-4132.
Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 Å resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m7G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.
doi:10.1093/nar/gkq122
PMCID: PMC2896518  PMID: 20194115
7.  A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI) 
Nucleic Acids Research  2010;38(9):3019-3030.
Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5′-GGCGC/C-3′ (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction–modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins.
doi:10.1093/nar/gkp1221
PMCID: PMC2875022  PMID: 20071747

Results 1-7 (7)