RlmCD belongs to the RFM family of MTases.

<p>(A) The canonical topology diagram of the catalytic domain in the RFM family of MTases. (B) Cartoon representation of the catalytic domain in RlmCD (residue 286–454). SAH is shown as ball-and-stick model. (C) The topology diagram of the catalytic domain in RlmCD. An extra α-helix (α5) is formed at the C-terminus of the catalytic domain.</p

Structural insights into substrate selectivity of ribosomal RNA methyltransferase RlmCD

<div><p>RlmCD has recently been identified as the S-adenosyl methionine (SAM)-dependent methyltransferase responsible for the formation of m⁵U at U747 and U1939 of 23S ribosomal RNA in <i>Streptococcus pneumoniae</i>. In this research, we determine the high-resolution crystal structures of apo-form RlmCD and its complex with SAH. Using an in-vitro methyltransferase assay, we reveal the crucial residues for its catalytic functions. Furthermore, structural comparison between RlmCD and its structural homologue RumA, which only catalyzes the m⁵U1939 in <i>Escherichia coli</i>, implicates that a unique long linker in the central domain of RlmCD is the key factor in determining its substrate selectivity. Its significance in the enzyme activity of RlmCD is further confirmed by in-vitro methyltransferase assay.</p></div

Overall structure of RlmCDs.

<p>(A) Three distinct parts of RlmCDs: the N-terminal TRAM domain, central domain, and C-terminal catalytic domain are colored in blue, green, and orange, respectively. The regions separating the three domains are all colored in grey. (B) The structure superimposition of RlmCDs and RumA (PDB ID 1UWV). RlmCDs and RumA are colored in gray and orange, respectively. (<i>Inset</i>) The superimposition of the central domain is individually shown to highlight the major difference between two structures. (C) The linker A and B of RlmCDs are shown in sticks as well as their electron density map with 2Fo-Fc calculated at 1σ.</p

The 23S rRNA helix 35 is the substrate of RlmCD.

<p>(A) Secondary structures of the 18-mer RNA fragments of the <i>S</i>. <i>pneumoniae</i> (left) and <i>E</i>.<i>coli</i> (right) 23S rRNA helix 35. (B) In-vitro methyltransferase assay of RlmCD. The left three columns represent the methyl transfer activities of the wild-type RlmCD or its mutants toward rRNA-h35. The right three columns represent the methyl transfer activities of the wild-type RlmCD toward the different derivatives of rRNA-h35 (U747A, U747G, and U747C).</p

SAH binds RlmCD at a canonical binding pocket.

<p>(A) The overview of SAH anchored onto the catalytic domain of RlmCD. RlmCD is shown in its electrostatic surface potential, and SAH is shown as ball-and-stick model. (<i>Inset</i>) A close-up of the engagement of SAH into the binding pocket. (B) The interaction details of SAH with RlmCD. RlmCD residues are colored in gray and SAH is colored in green. The gray mesh represents 2Fo-Fc calculated at 1σ density map of SAH and the dashed lines represent the hydrogen bonds.</p

RlmCD is a 23S rRNA methyltransferase.

<p>(A) Comparison of the MTase activities of wild-type RlmCD and its mutants using rRNA-h35 as the substrate. (B) Comparison of the MTase activities of RlmCD toward U747 and U1939. The MTase activity of wild-type RlmCD was normalized to 100%.</p

Dynamic Nature of CTCF Tandem 11 Zinc Fingers in Multivalent Recognition of DNA As Revealed by NMR Spectroscopy

The 11 zinc fingers (ZFs) of the transcription factor CTCF play a versatile role in the regulation of gene expression. CTCF binds to numerous genomic sites to form chromatin loops and topologically associated domains and thus mediates the 3D architecture of chromatin. Although CTCF inter-ZF plasticity is essential for the recognition of multiple genomic sites, the dynamic nature of its 11 ZFs remains unknown. We assigned the chemical shifts of the CTCF ZFs 1–11 and solved the solution structures of each ZF. NMR backbone dynamics, residual dipolar couplings, and small-angle X-ray scattering experiments suggest a high inter-ZF plasticity of the free-form ZFs 1–11. As exemplified by two different protocadherin DNA sequences, the titration of DNAs to ¹⁵N-labeled CTCF ZFs 1–11 enabled systematic mapping of binding of CTCF ZFs to various chromatin sites. Our work paves the way for illustrating the molecular basis of the versatile DNA recognized by CTCF and has interesting implications for its conformational transition during DNA binding

Structure of TbPRMT6.

<p>(A) Schematic diagram of the domain arrangement of TbPRMT6 (B) Overall structure of a monomer The N-terminal helix αY, the SAM-binding domain, the dimerization arm, and the β-barrel domain are shown in red, blue, yellow, and green, respectively The cofactor SAH is shown in the stick model The segment between helix αG and strand β7 is invisible and is shown as a dashed line (C) Structure of the TbPRMT6 dimer.</p

Dimerization of TbPRMT6.

<p>(A) Two views of the TbPRMT6 dimer Monomer A is shown as a cartoon colored in magenta, and monomer B is colored in gray and shown through a surface presentation (B) Dimerization interactions The left image represents the hydrogen bond interactions on the dimeric interface, and the right image represents the hydrophobic interactions (C) SAXS results of TbPRMT6_L The experimental SAXS curve of TbPRMT6 and the data points up to q  = 06 Å⁻¹ are plotted The top-right inset is the PDDF calculated by GNOM, and the bottom-left inset is the DR model with the crystal structure superimposed with a TbPRMT6 dimer The R_g and D_max of TbPRMT6 are 328±01 Å and 959 Å, respectively The DR models were generated by GASBOR using the final χ against the raw SAXS data of 063 The X-ray structure of the TbPRMT6 dimer can be superimposed onto the DR models quite well, with an NSD of 109.</p

Crystal Structure of Arginine Methyltransferase 6 from <i>Trypanosoma brucei</i>

<div><p>Arginine methylation plays vital roles in the cellular functions of the protozoan <i>Trypanosoma brucei</i>. The <i>T. brucei</i> arginine methyltransferase 6 (TbPRMT6) is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH). The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several <i>T. brucei</i> histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.</p></div