Fig. 3: Structural, kinetic and cellular analysis of targocil-II inhibition of S. aureus TarGH. | Nature Communications

Fig. 3: Structural, kinetic and cellular analysis of targocil-II inhibition of S. aureus TarGH.

From: Cryo-EM analyses unveil details of mechanism and targocil-II mediated inhibition of S. aureus WTA transporter TarGH

Fig. 3

a Chemical structure of targocil-II. b Inhibition of TarGH ATPase activity by targocil-II, as measured by a malachite green assay. Data points represent the mean from three independent trials ± standard deviation. Half-maximal inhibitory concentration (IC50) is reported as mean ± 95% confidence interval. c Microscale thermophoresis binding of AMP-PNP (left) or ATPγS (right) bound S. aureus TarGH to targocil-II. Experimental data is reported as the mean from at least three independent trials ± standard deviation and dissociation constant (Kd) reported as mean ± 95% confidence interval. d Cryo-EM reconstruction of targocil-II and AMP-PNP bound S. aureus TarGH (2.9 Å resolution). TarG is coloured in dark/light blue, TarH in dark/light green and targocil-II in dark orange. Targocil-II is circled. e View of the targocil-II binding site on TarG on left. Coloured as in (d). The cryo-EM density (map-threshold: 0.06) for targocil-II is depicted by a dark grey mesh. 2D chemical representation of binding site shown on right. Non-carbon atoms are coloured distinctly, and side chains are labelled with corresponding TarG structural feature. An asterisk (*) is used to denote positions of targocil-II resistance mutants. f Two-fold symmetric targocil-II binding sites at the extracellular portion of the TarG dimerisation interface with identified targocil and targocil-II resistance mutants coloured in grey. Targocil-II and interacting residues are shown as sticks with heteroatom colouring. TarG structural elements labelled. g Bacterial growth assay for gram-positive S. aureus, S. epidermidis, S. pneumoniae and B. subtilis strains in presence of targocil-II. Left: growth curve for inhibited strains. Cellular assay data from triplicate measurements is reported as the mean ± standard deviation. Right: table of minimum inhibitory concentrations (MIC). h Targocil-II binding site for S. aureus (left; experimental) and S. epidermidis (right; AlphaFold model). Residues defining the binding site shown as spheres with differences at position 53 (Met vs Leu), 191 (Phe vs Met) and 203 (Ile vs Val) around the chlorophenyl binding pocket.

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