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. 2020 Nov 20;16(11):e1009016. doi: 10.1371/journal.ppat.1009016

Fig 2. Loss of an essential cation-π interaction inhibits transmembrane β-hairpin (TMH) formation in Ply-NH.

Fig 2

(A) Cartoon representation of Ply-NH crystal structure where individual domains are labelled as D1, D2, D3 and D4. The substitutions are represented as spheres along with ball and stick and colored according to elements (C-cyan, N-blue and O-red). The deletion is marked by black arrow. The TMH1 and TMH2 are also highlighted in blue and brown, respectively. (B) Electrostatic surface representation of Ply-NH. The blue and red indicate the electropositive and electronegative regions, respectively. (C) Multiple sequence alignment of CDCs highlighting the conservation of tyrosine (highlighted in yellow, Y150 in Ply-H) and phenyl alanine (highlighted in magenta, F318 in perfringolysin). (D) Hemolytic activity of Ply-H and its mutated variants (E286A, V287A and K288A) at different concentrations for identification of the potential β4 residue which pairs with Y150 from β1 of neighbouring monomer. (E) Cartoon representation of extended TMHs of two Ply-H/Ply-NH monomers (M1 and M2 colored cyan and green for Ply-H and brown and grey for Ply-NH, respectively) in the oligomerized state. Inset: Zoomed in view showing inter-molecular cation-π interaction between K288 and Y150 in Ply-H and its disruption in Ply-NH with H150 substitution. (F) Close up view of the hydrophobic pocket in domain-3 formed by F169, I172, L176, Y247, V288 and L290 residues, which are shown as both sphere and ball and stick style (wheat color). The I172 (ball and stick model in red color) is found to be stabilized in the hydrophobic pocket. (G) Concentration dependent hemolytic profile of Ply-NH and its mutants showing gain of hemolytic activity. Individual mutations H150Y and I172T in Ply-NH show some gain of hemolytic activity, notably the double mutant (H150Y+I172T) shows gain of most of the hemolytic activity. Data information: Experiments are performed thrice and data of representative experiments are presented as mean ± SD of triplicate wells (D, G). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test vs Ply-H (D) or Ply-NH (G). ns, non-significant; ***p<0.001.