Fig 1.
STD, WATERlogsy and CPMG ligand-detected experiments carried out on Fragment 2 in the absence (green traces) and presence (orange traces) of FtsYNG.
The top (black) trace is the 1H 1D NMR spectrum of Fragment 2 for reference. (B−D) 15N-TROSY-HSQC spectrum of 15N-FtsYNG alone (blue) and following addition of (B) Fragment 2 (red), (C) GTP analogue (red) and (D) 4.5S RNA (red). Arrows indicate the peaks that have shifted during the titration with green arrows highlighting the same peaks that have shifted in the fragment titration and the 4.5S RNA titration. (E) Chemical structures of Fragments 1, 2 and 3.
Fig 2.
In vitro and in vivo validation of fragments.
SPR measurements performed after immobilization of FtsYNG in sensor chip and Fragment 1 (A), Fragment 2 (B) and Fragment 3 (C) were injected at (6.25–200 μM). As a positive control GMPPNP (D) was injected (6.25 μM–400 μM) and their KD determined. E) Zone inhibition assays using E.coli BL21were performed with 150 μg of Fragment 1, 2 or 3 spotted in a filter paper prior to overnight incubation at 37°C. Kanamycin (30 μg) was used as a positive control.
Fig 3.
(A) FtsYNG bound to Fragment 1 (shown in green) in the Trp343 and Phe365 binding sites. (B and C) FtsYNG:Fragment 1 interaction profile in (B) Trp343 binding site and (C) Phe365 binding site. (D) FtsYNG bound to Fragment 2 (shown in orange) in the Trp343 and Phe365 binding sites. (E and F) FtsYNG:Fragment 2 interaction profile in (E) Trp343 binding site and (F) Phe365 binding site. (G) FtsYNG bound to Fragment 3 (shown in pink) in the Trp343 and Phe365 binding sites. (H and I) FtsYNG:Fragment 3 interaction profile in (H) Trp343 binding site and (I) Phe365 binding site. Fragments are displayed as sticks with Fragment 1 shown in green, Fragment 2 in orange, Fragment 3 in pink and the amino acids interacting with them shown as blue sticks. Interactions are water bridge (grey line), hydrophobic (red dashed line), hydrogen bond (blue line) and π-stacking (green dashed line). Distances for interactions are indicated in the figure. mFo-DFc fragment electron density map is shown in grey and contoured at 3σ level in (A), (D) and (G).
Fig 4.
Chemical environment surrounding the fragment-binding sites.
In Trp343 binding site: (A) Fragment 1 (green), (B) Fragment 2 (orange) and (C) Fragment 3 (magenta). (D−F) In Phe365 binding site: (D) Fragment 1, (E) Fragment 2 and (F) Fragment 3. FtsYNG is shown as surface with amino acids that form the surrounding fragment binding site labelled and shown as blue sticks. Fragment 1 is shown in green, Fragment 2 in orange and Fragment 3 in magenta. R represents positions to be modified according to the Phe365 binding site.