Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis

Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH3). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N6-isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms2i6A formation (MiaB activity), and yqeV is required for modification of N6-threonylcarbamoyladenosine (t6A) to 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i6A from t6A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis

Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis

Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH3). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N6-isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms2i6A formation (MiaB activity), and yqeV is required for modification of N6-threonylcarbamoyladenosine (t6A) to 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i6A from t6A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis

Cellular dynamics of tRNAs and their genes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116361/1/feb2s0014579309009661.pd

Identification of STAT1 and STAT3 specific inhibitors using comparative virtual screening and docking validation.

Signal transducers and activators of transcription (STATs) facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr)-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (h)STATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the 'STAT-comparative binding affinity value' and 'ligand binding pose variation' as selection criteria. In silico screening of a multi-million clean leads (CL) compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our understanding of the functional role of these STATs in different diseases and benefit the clinical need for more drugable STAT inhibitors with high specificity, potency and excellent bioavailability

Binding conformations of top-scored compounds from natural products library in the SH2 domain of hSTAT1 and hSTAT3.

<p><b>(A)</b> Binding pose variation of the top-scored hSTAT1-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. <b>(B)</b> Binding pose variation of the top-scored hSTAT3-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. The binding pose variations are shown in line representation, colored in blue and violet. Results were obtained using Surflex-Dock 2.6 program.</p

STAT3-CBAVs of STAT3-specific inhibitors.

<p>Graph presents comparative binding affinity values of a selection of STAT3-specific inhibitors docked to models of all hSTAT monomers.</p

Structural models and phylogenetic comparison of hSTAT monomers (1, 2, 3, 4, 5A, 5B and 6) with their specific pTyr-linkers.

<p><b>(A)</b> Phylogenetic distribution of hSTATs in form of a simplified phylogenetic tree. <b>(B)</b> Models of the monomers are shown in the cartoon representation with pTyr-peptides in the stick representation. Specific domains are positioned as follows: N-domain on the top-left, coiled-coiled domain on the bottom-center, C-domain on the top-right and SH2 domain on the top-center, to facilitate visual analysis of phosphotyrosine (pTyr)-linker and the SH2 interactions. Monomers are colored according to the predicted local deviation from the real structure (the predicted error of the model), as calculated by MetaMQAP. Blue indicates low predicted deviation of Cα atoms down to 0Å, red indicates unreliable regions with deviation > 5Å, green to orange indicate intermediate values. pTyr-peptides are colored in violet, while pTyr residue is colored in pink. <b>(C)</b> Models of hSTAT dimers with the linker of monomer I in the SH2 domain of monomer II. pTyr-peptides are presented in stick representation, pY+0—pTyr binding pocket, pY-X—hydrophobic side-pocket. SH2 domains are in the surface representation, colored according to the distribution of the electrostatic surface potential, calculated with APBS. Blue indicates positively charged regions, red indicates negatively charged regions.</p

Binding conformations of top-scored compounds from clean leads library in the SH2 domain of hSTAT1 and hSTAT3.

<p>(A) Binding pose variation of the top-scored hSTAT1-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. (B) Binding pose variation of the top-scored hSTAT3-specific inhibitor in SH2 domain of hSTAT1 and hSTAT3. The binding pose variations are shown in line representation, colored in yellow and green. Results were obtained using Surflex-Dock 2.6 program.</p

Top-scored binding conformation of stattic in the SH2 domain of all hSTATs.

<p>Stattic is shown in stick representation, pTyr-linker is presented as green colored lines with pTyr residue in pink. Results were obtained using Surflex-Dock 2.6 program.</p