Fig. 3: Ca²⁺ binds plectasin and increases lipid II affinity.

a, 2D NH solution NMR spectra of unbound plectasin in the absence (blue) and the presence (yellow) of paramagnetic Mn²⁺. b, Signal intensities derived from the NMR spectrum in a in the presence of Mn²⁺ show that Mn²⁺ binds the anionic patch and the β-loop. Signals marked with asterisks (*) were not assigned. a.u., arbitrary unit. c, Illustration of the ion binding site. Ca²⁺ was manually modelled. The insert shows a ⁴³Ca ssNMR spectrum of Ca²⁺ (in magenta) bound to the plectasin–lipid II complex in DOPG membranes; the control spectrum (in black) in DOPG membranes was recorded in the absence of plectasin; a spectrum of free Ca²⁺ in buffer is shown in cyan. d, Plectasin to lipid II binding affinities measured by tryptophan fluorescence (left) in zwitterionic DOPC membranes and (right) in anionic DOPC/DOPG membranes (1:1 mixture) with 1 mM Ca²⁺ (magenta), 1 mM Mg²⁺ (green) and in the absence of bivalent ions (100 μM EDTA, grey). Source data are provided. e, Dipolar ssNMR data show a rigidification of the anionic side chains E10 and D12 of lipid II-bound plectasin in the presence of Ca²⁺, confirming that bivalent ions bind to the complex. f, Side chains D9/D11 define the conformation of the N-loop, while E10/D12 bind to Ca²⁺. g, Zooms into 2D PARISxy⁴⁵ CC ssNMR data show that Ca²⁺ binding allosterically modulates the αβ-loop (I22) and the β-sheet (Y29), in red without Ca²⁺ and in blue with Ca²⁺. h, ssNMR ¹⁵N T_1ρ dynamics show that Ca²⁺ rigidifies plectasin at sub-stoichiometric plectasin to lipid II concentrations. The error bars show the standard error of the fit. Source data are provided.

Source data