Skip to main content
. 2020 May 29;9:e56048. doi: 10.7554/eLife.56048

Figure 6. Crystal structure of ReoM.

(A) The structure of ReoM depicted as a cartoon with each protomer in the dimer coloured separately (cyan and orange). The secondary structure elements are numbered according to their position in the amino acid sequence. Thr7 and some of the key amino acids in the dimer interface and the hydrophobic core are drawn as stick figures. (B) Sequence alignment of ReoM (Lmo) and its homologues from Bacillus subtilis (Bsu), Streptococcus pneumoniae (Spn), Clostridium difficile (Cdi) and Staphylococcus aureus (Sau) with the sequence of IreB from Enterococcus faecalis (Efa) underneath. Amino acid sequence numbers pertain to ReoM and the site of phosphorylation in ReoM (Thr7) and the twin phosphorylations in IreB (minor site: Thr4; major site: Thr7) are highlighted. Invariant amino acids are shaded black, residues in the ReoM dimer interface have an asterisk above, and the secondary structure elements are defined by cylinders above the alignment. Arginine residues mutated in this study are indicated by a hashtag above the alignment. (C) The final 2Fobs-Fcalc electron density map, contoured at a level of 0.42 e-/Å3, of the N-terminal region in the immediate vicinity of Thr7 in chain A of the ReoM dimer indicates that the protein model could be built with confidence even though this region contains no secondary structure elements.

Figure 6.

Figure 6—figure supplement 1. ReoM and P-ReoM have the same oligomeric state.

Figure 6—figure supplement 1.

Size exclusion chromatography of ReoM and P-ReoM showed that phosphorylation of ReoM does not alter its oligomeric state. Both ReoM (solid) and P-ReoM (dashed) had an elution volume at 14.6 mL.
Figure 6—figure supplement 2. Lethality of ReoM R57A and R62A substitutions.

Figure 6—figure supplement 2.

L. monocytogenes strains EGD-e (wt), LMSW52 (ireoM T7A), LMSW125 (ireoM R57A), and LMSW126 (ireoM R62A) were grown in BHI broth ±1 mM IPTG at 37°C. The experiment was repeated three times and average values and standard deviations are shown.
Figure 6—figure supplement 3. A possible conformational change of the flexible ReoM N-terminus induced by phosphorylation.

Figure 6—figure supplement 3.

The N-terminus of ReoM might undergo a substantial movement after phosphorylation that would be stabilised in its new conformation by electrostatic interactions between the negatively charged phosphate of Thr7 ~P and the positively charged Arg57/Arg62 pair.