Table 1.
Data collection and refinement statistics.
Fig 1.
Overall structure of RsmI-AdoMet complex.
(A) Cartoon representation showing the domain architecture of RsmI with NTD in cyan and CTD in yellow. The binding AdoMet molecule is shown in green sticks. (B) Dimeric structure of RsmI, with subunit I and II shown in cartoon and surface, respectively. The NTD and CTD of subunit II are shown in light and dark gray, respectively. The two subunits make contacts by a “back to back” mode and the AdoMet-binding site is exposed. (C) Structure-based sequence alignment for representative RsmI family members from gram negative and positive bacteria, including E. coli, Pseudomonas aeruginosa (P_ aeruginosa), Rickettsia prowazekii (R_prowazekii), Helicobacter pylori (H_pylori), Mycobacterium tuberculosis (M_tuberculosis), Staphylococcus aureus (S_aureus) and Mycoplasma pneumoniae (M_pneumoniae), performed using clustal X (version 1.81) and ESPript 3.0 [31]. The conserved residues are boxed in blue. Identical conserved and low conserved residues are highlighted in red background and red letters, respectively. The residues involved in RsmI dimer formation are labelled using stars and those involved in AdoMet-binding are labelled using triangles.
Fig 2.
Structure comparisons of RsmI with its homologs.
(A-B) Structural superimpositions of RsmI (green) with representative members of AdoMet-dependent MTases involved in cobalamin biosynthesis, including CbiF (cyan, PDB code 1CBF) and CbiL (orange, PDB code 2E0N). One phosphate molecule (orange sticks) is bound to the proposed substrate binding site in NRD that is close to the AdoHcy molecule in CbiF. (C) Structural superimpositions of AdoMet (green) with AdoHcy in CbiF (cyan) and CbiL (orange), showing similar bent conformations in class III MTases family. (D) Structural superimpositions of RsmI NTD (cyan) with representative members of protein tyrosine phosphatase (PTP) superfamily, PRL-1 (wheat, PDB code 1XM2) and PIR1 (pink, PDB code 4NYH). The active-site bound sulfate ion (orange sticks) in PTPs is known to mimic the substrate phosphate group. (E) Structural superimpositions of RsmI CTD (yellow) with rRNA MTases RlmE (gray, PDB code 1EIZ) and RlmM (purple, PDB code 4B17). RsmI CTD adopts a Rossmann-like fold distinguished from those of RlmE and RlmM that belong to class I MTase family.
Fig 3.
AdoMet binding in the active site formed by NTD and CTD.
(A) Contacts analysis between AdoMet (green sticks) and RsmI (gray cartoon). The interacting residues from NTD and CTD are highlighted using cyan and yellow sticks, respectively. A water molecule (W) is shown as red sphere. Electron density map (2Fo-Fc) of AdoMet is contoured at a 2σ level. (B) AdoMet binds into a deep pocket formed by several flexible loops from both NTD and CTD (shown as surface electrostatic potential, blue, +7.8KT; red, -7.8KT), with dominantly negative charges.
Table 2.
ITC data for titration of RsmI variants with AdoMet and putative substrates fitting using a one-site binding model.
Fig 4.
ITC spectra for the binding of RsmI wild-type and mutants to AdoMet/cytidine/CMP.
Baseline subtracted raw ITC data for injections of AdoMet (cytidine or CMP) is indicated in the upper panels of each of the ITC profiles shown (for the wild-type as well as the variants of RsmI). The peaks normalized to the ligand/protein molar ratio were integrated as is shown in the bottom panels. The solid dots indicate the experimental data and the best fit to the experimental data were obtained from a non-linear least squares method of fitting using a one-site binding model depicted by a solid line. The binding affinity of each mutant is from the average of three independent experiments.
Fig 5.
Docking model of RsmI-AdoMet-rRNA ternary complex.
(A) A molecular surface and cartoon representation of RsmI, colored by its local electrostatic potential. The substrate RNA is bound into the cleft between NTD (in cyan) and CTD (in yellow) and close to AdoMet (green sticks). AdoMet and C1402 are shown in green and magenta sticks, respectively. (B) A detailed view of the interactions in the model. Arg174 (yellow sticks) can form an H-bond with the phosphate backbone linking C1402. The distance between the AdoMet methyl group and the target O2’ atom of C1402 is 4.7 Å, which may require a slight shift to trigger catalysis.