Mode of action of teixobactins in cellular membranes

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.

The high spectral quality and the absence of peak-doubling (expect for a second conformation for the Ile6 sidechain) demonstrate that the complex is well-defined. The spectrum is fully annotated. b) Site-resolved 15 N R 1rho ssNMR dynamics of Lysine-Lipid II-bound [R4L10]teixobactin acquired at 60 kHz magic angle spinning (MAS) in DOPC liposomes. The size of the magenta spheres illustrates the dynamics. Relaxation in the entire molecule is very slow, even slower than in membrane-embedded α-helices of ion channels. 1 The very slow relaxation strongly suggests that the N-terminus is immobilised in a supramolecular interaction. Residues without 13 C, 15 N-labels are marked by green stars. The error bars show the standard error of the fit. Source data are provided as a Source Data file. c) Superposition of 2D NH spectra of free [R4L10]-teixobactin in aqueous buffers (in red) and of the Lipid II-bound state in DOPC membranes (cyan). The strong spectral changes demonstrate a well-defined interaction with Lipid II.

Supplementary Figure 3
Pyrene excimer fluorescence shows that clustered Lipid II molecules bound to teixobactins are within 1 nm of each other in DOPC vesicles.
Monomers of pyrene exhibit characteristic fluorescence emission maxima at 378, 398, and 417 nm. Moreover, pyrene can display a characteristic fluorescence peak at longer wavelengths (490 nm), which occurs only when two pyrene rings reside within 10 Å of each other and form an excited state dimer, which known as an excimer. 2 Source data are provided as a Source Data file. a) Fluorescence spectra of pyrene-labelled Lipid II symmetrically in large unilamellar vesicles (LUVs) at increasing concentrations of [R4L10]-teixobactin. The spectra show that the fluorescence of the Lipid II monomers goes down while the excimer fluorescence increases. The pyrene fluorescence experiments suggest that the dimerization of teixobactins is almost immediate, and that the oligomerisation into clusters follows from this dimerization. b) The insert shows the excimer region of plot a). c) Quantification of the excimer over monomer ratio as a function of the pyrenelabelled Lipid II concentration. Supplementary Figure 6 The pentapeptide of Lipid II is mobile in the complex. Experiments that rely on so-called scalar couplings can be used to detect residues that show fast nanosecond motion with large amplitudes. I.e., scalar experiments are complementary to experiments that rely on so-called dipolar couplings and which only show rigid residues. a) Zoom into a scalar 2D 13 C 13 C TOBSY (TOtal through Bond correlation SpectroscopY) 4,5 ssNMR spectrum of the complex formed by 13  Conversely, the last three residues (Lys3-Ala4-Ala5) of the Lipid II pentapeptide give strong scalar signals, while γ-Glu2 shows gives clear but weaker scalar signals. Ala1, which is directly bound to the rigid MurNAc, is only visible in dipolar-based spectra (see Fig. 2d of the manuscript). b) A larger spectral region of the 2D 13 C 13 C TOBSY spectrum. No sugar signals could be detected, demonstrating that both MurNAc and GlcNAc sugars are rigid in the complex. c) The mobility profile of the pentapeptide of [R4L10]-teixobactin-bound Lipid II.
In our TOBSY sequence, the basic recoupling pulse cycle (90 x -360 -x -270 x ) needs to be repeated three times during one rotor period in order to suppress anisotropic NMR interactions. This led to a 13 C power requirement of six times the MAS frequency. Therefore, we used slow MAS (8 kHz) for this experiment. Figure 7 [R4L10]-teixobactin localises at the water-lipid interface while hydrophobic sidechains act as membrane anchors. a) and b) show motion-edited 1 H( 1 H) 13 C ssNMR experiments. 6,7 In these experiments, application of a T 2 -relaxation filter destroys magnetization of the rigid complex while magnetization on mobile water and lipid molecules is maintained and subsequently transferred to the rigid Lipid II-bound [R4L10]-teixobactin via 1 H-1 H mixing. The magnetization is eventually transferred to the 13 C nuclei of the rigid antibiotic via a short (200 us) dipolar cross polarisation step. A 2D implementation of this experiment enables to measure to which extent residues partition into water and lipid phases. The 2D experiment unambiguously shows that the sidechains of Ile2 and Ile6 partition into the lipid phase. All spectra were measured at 700 MHz ( 1 H-frequency), 16.5 kHz MAS, and at 300 K sample temperature.

Supplementary
a) 1D mobility-edited 1 H( 1 H) 13 C ssNMR spectrum of [R4L10]-teixobactin bound to Lipid II measured at 700 MHz using a T 2 relaxation delay of 2.5 ms to de-phase signals of the complex. 1 H-1 H mixing was set to zero, i.e., no magnetization was transferred to the rigid complex, and all visible signals relate to residual lipid signals, demonstrating the effectiveness of the T 2 mobility filter. The spectrum was acquired with 10240 scans. b) 2D mobility-edited 1 H( 1 H) 13 C ssNMR spectrum of [R4L10]teixobactin bound to Lipid II measured at 700 MHz using a T 2 -relaxation filter of 2.5 ms. Subsequently, 5 ms 1 H-1 H mixing was used to transfer magnetization from mobile water and lipid tails to the complex. The 2D spectrum was measured with 8192 scans. c) 13

Supplementary Figure 9
Interactions on the water-exposed part of the β-sheet formed by Lipid II-bound [R4L10]-teixobactin. a) The long hydrophobic sidechains of D-N-Me-1, Ile2, D-allo-Ile5, and Ile6 act as essential membrane anchors, whose replacement is not tolerated. [8][9][10] Ile11 is waterexposed in the complex, and its replacement by alanine is tolerated. Weaker binding affinity to Lipid II in anionic conditions is a common property of the binding mode of teixobactins. The spectra were acquired at 700 MHz with 60 kHz magic angle spinning (MAS).

NMR restraints and Structure validation
Intramolecular [R4L10]-teixobactin distance restraints: We defined intramolecular restraints between residues Ser7 to residues Ala9 and Leu10 based on 2D 13 C 13 C PARIS experiments with different magnetization transfer times using upper and lower limits of 7.0 and 2.0 Å, respectively (8.0 and 3.0 Å for few weak signals).
Dihedral angle restraints (intramolecular): Dihedral angle restraints were applied for Ile2-Ile7 of [R4L10]-teixobactin. Since the N-terminus of [R4L10]-teixobactin, [L10]teixobactin, and natural teixobactin 14,15 form β-sheet structures, we extracted average dihedral angle from the X-ray structure of a teixobactin in complex with a sulphate ion, where the N-terminus has β-strand conformation. 16 Restraints were implemented with boundaries of +/-20°. We validated this procedure by deriving dihedral angles from our ssNMR assignments using the TALOS+ 17 software. Indeed, ssNMR-based dihedrals could be obtained for residues Ile2 and Ile6 (with 'Good' accuracy, i.e., the best accuracy), which matched well to the N-terminus of the X-ray structure.
Intramolecular Lipid II distance restraints: Contacts within and between the MurNAc and GlcNAc sugar were defined with upper and lower limits of 6.0 and 1.5 Å, respectively, based on a series of 2D 13 C 13 C PARIS.

Intermolecular distance restraints between [R4L10]-teixobactin molecules
All restraints based on a series of 2D 13 C 13 C PARIS experiments using upper and lower limits of 7.0 and 2.0 Å, respectively (8.0 and 3.0 Å for few weak signals). These restraints were fulfilled in all 26 structures.

[R4L10]-teixobactin C with [R4L10]-teixobactin D Similar as in A with B
Intermolecular distance restraints between [R4L10]-teixobactin and Lipid II: Restraints involving the pyrophosphate group: Ambiguous distance restraints were applied between the backbone amino protons of the four ring residues (Thr8-Ile11) with either phosphate of the pyrophosphate group using upper and lower limits of 2.4 and 1.7 Å, respectively. Restraints based on a 2D 1 H 31 P experiment (Fig. 2b of the  manuscript). Restraints involving the MurNAc and GlcNAc sugars: Restraints were applied using boundaries of using upper and lower limits of 8.0 and 1.5 Å, respectively (9.0 and 2.0 Å for few weak signals). Restraints were based on a series of 2D 13 C 13 C PARIS experiments Topological restraints: Eventually, a filtering strategy was applied. Structures were only accepted if all Lipid II tails pointed into the direction of the membrane-anchoring residues Ile2, Ile5, and Ile6) (see Fig. 2f of the main text and Supplementary Figure  7). Sorting of the Lipid II tails was steered by imposing distance restraints between the sidechain of Ile6 and the Lipid II isoprenyl-tail.
Synthesis of 13 C, 15  Amino acids in red denote 13 C and 15 N labeled atoms.
(step a) Commercially available 2-Chlorotrityl chloride resin (manufacturer's loading = 1.6 mmol/g, 150 mg resin) was swelled in DCM in a reactor. To this resin was added 4 eq. Fmoc-Ala-OH/8 eq. DIPEA in DCM and the reactor was shaken for 3h. The loading determined by UV absorption of the piperidine-dibenzofulvene adduct was calculated to be 0.56 mmol/g, (150mg resin, 0.084 mmol). Any unreacted resin was capped with MeOH:DIPEA:DCM = 1:2:7 by shaking for 1h. (step b) The Fmoc protecting group was deprotected using 20% piperdine in DMF by shaking for 3 min, followed by draining and shaking again with 20% piperidine in DMF for 10 min. AllocHN-D -Thr-OH was then coupled to the resin by adding 3 eq. of the AA, 3 eq. HATU and 6 eq. DIPEA in DMF and shaking for 1.5h at room temperature. (step c) Esterification was performed using 10 eq. of Fmoc-Ile-OH, 10 eq. DIC and 5 mol% DMAP in DCM and shaking the reaction for 2h. This was followed by capping the unreacted alcohol using 10% Ac 2 O/DIPEA in DMF shaking for 30 min and Fmoc was removed using protocol described earlier in step (b). (step d) Fmoc-Leu-OH was coupled using 4 eq. of AA, 4 eq. HATU and 8 eq. DIPEA in DMF and shaking for 1h followed by Fmoc deprotection using 20% piperidine in DMF as described earlier.
(step e) The N terminus of Leu was protected using 10 eq. Trt-Cl and 15% Et 3 N in DCM and shaking for 1h. The protection was verified by the Ninhydrin colour test.
(step f) The Alloc protecting group of D-Thr was removed using 0.2 eq. [Pd(PPh 3 )] 0 and 24 eq. PhSiH 3 in dry DCM under argon for 20 min. This procedure was repeated again increasing the time to 45 min and the resin was washed thoroughly with DCM and DMF to remove any excess Pd stuck to the resin. (step g) All amino acids were coupled using 4 eq. Amino Acid, 4 eq. HATU and 8 eq. DIPEA for 1hr. (step h) The peptide was cleaved from the resin without cleaving off the protecting groups of the amino acid sidechains using TFA:TIS:DCM = 2:5:93 and shaking for 1h. (step i) The solvent was evaporated and the peptide was dissolved in DMF to which 1 eq. HATU and 10 eq. DIPEA were added and the reaction was stirred for 30 min to perform the cyclisation. (step j) The side-chain protecting groups were then cleaved off using TFA:TIS:H2O = 95:2.5:2.5 by stirring for 1h. The peptide was precipitated using cold Et 2 O (-20°C) and centrifuging at 7000 rpm to obtain a white solid. This solid was further purified using RP-HPLC using the protocols described previously (Supplementary Figure 16c,d). 19 The teixobactin analogues (1-2) were identified by MS in positive mode (Supplementary Table T4  Supplementary Figure 16| Characterisation of labelled amino acids (compounds S1 & S2) and labelled peptides (analogues 1 & 2). a) 13 C NMR Spectra of compound S1. b) 13  Syntheses of 13 C, 15 N-labelled Fmoc-amino acids: Supplementary Figure 17| Syntheses of Fmoc protected labelled amino acids, S1 and S2 1eq. of labeled L -amino acid (Serine/Isoleucine) was dissolved in 4eq of NaHCO 3 in water. Subsequently, a solution of 1.2eq. Fmoc-OSu in THF was added dropwise to the stirring solution of amino acid at room temperature (rt) for 30min. The reaction was further stirred overnight at room temperature. The THF was evaporated under reduced pressure and the aqueous layer was washed with diethyl ether (Et 2 O). The aqueous layer was acidified using 2M HCl (pH 2) and then extracted with ethyl acetate (EtOAc) twice. The organic phases were combined and further washed with brine and treated with anhydrous sodium sulphate (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure. The white amorphous solid of Fmoc-amino acids yielded quantitatively and was used without purification.