Production of LptA-His6, LptAm, LptD/E, and thanatin in E. coli and details of binding assays with LptA by FP and thermophoresis are described in sections S7 to S9. Production of [15N]- and [15N,13C]-labeled LptAm was performed in BL21(DE3) E. coli cells grown in M9 minimal medium appropriately supplemented with 15NH4Cl and 13C glucose at 25°C overnight. Purification is described in section S7. The addition of 20 mM CHAPS to the sample buffer significantly improved the quality of the [15N,1H]-HSQC spectra by reducing line-broadening effects due to aggregation (see figs. S8 and S9). Final NMR samples contained 50 mM sodium phosphate, 150 mM NaCl (pH 7.5), and protein/peptide concentrations of 0.5 to 0.6 mM.

NMR spectra were acquired at 290 K (free 15N-thanatin) and at 308 K (free 15N,13C LptAm, and complex of LptAm-thanatin) using 700- and 600-MHz Bruker NEO spectrometers. All spectra were processed using TopSpin 4.0 and analyzed using CARA and CCPNmr. The 1H, 15N, and 13C chemical shifts of backbone and side-chain atoms were assigned using a standard set of triple-resonance experiments on either uniformly 15N,13C-labeled LptAm with or without unlabeled thanatin or with uniformly 15N,13C-labeled thanatin with or without unlabeled LptAm at protein concentrations of 0.5 to 0.6 mM. The LptAm-thanatin complex was prepared at a ratio of 1:1.2.

Backbone assignment was initiated from manually picked [15N,1H]-HSQC spectra that served as anchoring points for HNCO, HN(CO)CACB, and HNCACB experiments (34). Sequential resonance assignments used the standard strip matching procedure for Cα/Cβ chemical shifts. Backbone and side-chain chemical shift assignments were obtained for 89.7 and 85.7% of residues 28 to 143 of LptAm and 92.7 and 96.6% of residues 1 to 21 of thanatin, respectively. We noticed that resonances from the presumably unstructured C-terminal tail including the His tag (residues 144 to 170) and some residues in the longer loop regions (β6-β7) were often missing, likely because of accelerated amide proton exchange at pH 7.5. To this end, we adjusted the sample of the LptA-thanatin complex to pH 4.6 and remeasured the triple-resonance spectra. The overall signal dispersion in the [15N,1H]-HSQC spectra was not changed significantly, indicating a stable complex formation under those conditions. Additional amide cross peaks could be assigned to residues located in solvent-exposed loops or strands, namely, Gly30, Gln43, Met47, Gly78, Asp101, and Asp139. Many more peaks became visible that were often characterized by negative values of the 15N{1H}-NOEs, indicating that they belonged to flexible amide moieties but could not be assigned unambiguously.

Hβ and Hα chemical shifts obtained from the HBHA(CO)NH experiment were used in combination with Cα/Cβ chemical shifts from the backbone assignments to obtain side-chain assignments in HCCH experiments. The aromatic side chains were linked to the backbone using the (HB)CB(CGCDCD)HD and (HB)CB(CGCDCDCE)HE experiments (35). The assignment of thanatin resonances in the complex was performed in a similar manner. Proton chemical shifts were referenced to the water line at 4.65 ppm at 308 K, from which the nitrogen and carbon scales were derived indirectly by using the conversion factors of 0.10132900 (15N) and 0.25144954 (13C). All chemical shifts were deposited in the Biological Magnetic Resonance Data Bank (BMRB) database under ID 34261.

Upper-distance restraints used for the structure calculations of the LptAm-thanatin complex were generated from 70-ms 15N- and 13C-NOESY (aliphatic and aromatic carbons) spectra. Intermolecular restraints were obtained from 70-ms 13C,15N-filtered/13C-edited (aliphatic and aromatic 13C), and 13C,15N-filtered/15N-edited NOESY spectra, and all experiments were performed on two samples (13C,15N-labeled LptAm/unlabeled thanatin and unlabeled LptAm/13C,15N-labeled thanatin). Additional torsion angle restraints were derived from backbone chemical shifts using the program TALOS+ (36). The solution structure of the LptAm-thanatin complex was determined using distance restraints derived from a set of NOESY spectra and torsion angle restraints derived from TALOS+. A full description of the structure calculations and statistical analysis of results is given in the Supplementary Materials.

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