High-resolution NMR studies of antibiotics in cellular membranes

Abstract

The alarming rise of antimicrobial resistance requires antibiotics with unexploited mechanisms. Ideal templates could be antibiotics that target the peptidoglycan precursor lipid II, known as the bacterial Achilles heel, at an irreplaceable pyrophosphate group. Such antibiotics would kill multidrug-resistant pathogens at nanomolecular concentrations without causing antimicrobial resistance. However, due to the challenge of studying small membrane-embedded drug–receptor complexes in native conditions, the structural correlates of the pharmaceutically relevant binding modes are unknown. Here, using advanced highly sensitive solid-state NMR setups, we present a high-resolution approach to study lipid II-binding antibiotics directly in cell membranes. On the example of nisin, the preeminent lantibiotic, we show that the native antibiotic-binding mode strongly differs from previously published structures, and we demonstrate that functional hotspots correspond to plastic drug domains that are critical for the cellular adaptability of nisin. Thereby, our approach provides a foundation for an improved understanding of powerful antibiotics.

DNP‐Supported Solid‐State NMR Spectroscopy of Proteins Inside Mammalian Cells

“…For comparison, Figure 4a lso includes results of a2 DD NP-ssNMR experiment on Ub before delivery into cells (black) in DNP buffer and in vitro NMR assignments of Ub at ambient temperature (black crosses,see Section 3.16 of the Supporting Information). To further improve spectral resolution, we resorted to 3D ssNMR (2Q-1Q-1Q) 13 C, 13 Cspectroscopy [26] (Figure 5and Supporting Information, Figures S9-S12). To further improve spectral resolution, we resorted to 3D ssNMR (2Q-1Q-1Q) 13 C, 13 Cspectroscopy [26] (Figure 5and Supporting Information, Figures S9-S12).…”

“…[1][2][3][4] On the other hand, solid-state NMR spectroscopy (ssNMR) has been used to probe proteins and large protein complexes in bacterial cells [5][6][7][8][9] and at the cell membrane periphery of human cells. Dynamic nuclear polarization (DNP [11] ), in which polarization is transferred from free electrons to atomic nuclei, greatly enhances ssNMR sensitivity.P revious work has shown that DNP is readily compatible with ssNMR on bacterial [7a] and human [10,12] cells,c ell compartments [7][8][9][10]13] as well as cell lysates. Furthermore,cellular ssNMR studies should be possible at endogeneous protein concentrations to ensure proper cell functioning.A sar esult, highsensitivity ssNMR methods are needed that allow such proteins to be studied in an intact cellular environment.…”

BceAB-Type Antibiotic Resistance Transporters Appear To Act by Target Protection of Cell Wall Synthesis

“…In the case of BceAB, a shift in equilibrium from target-bound AMP to free AMP can be achieved if the transporter has a low affinity to the free antibiotic and therefore releases it as soon as it is removed from the target. Given the substantial conformational change of bacitracin and other peptides between the free and target-bound forms (1,(33)(34)(35), this seems entirely plausible. In support of this idea, we have only been able to show binding of bacitracin by the entire detergent-solubilized transporter, where UPP may have been copurified (6), but never with its isolated extracellular domain that provides substrate specificity in BceAB-like systems (36) and therefore is thought to contain the ligand-binding site (our unpublished data).…”

Nucleotide Binding Modes in a Motor Protein Revealed by 31P‐ and 1H‐Detected MAS Solid‐State NMR Spectroscopy

“…These interactions have been typicallys tudied in the past by high-resolution X-ray crystallography. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies. [8] And indeed, solid-state NMR spectroscopy has been used to identify residues at protein-RNA interfaces in smaller proteins.…”

“…[9] Intermolecular information can also be obtained from 31 P-detected, heteronuclear correlation experimentsp robingt he spatial proximity of nucleotide 31 Pa nd protein 15 No r 13 Cn uclei. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] buta lso in experimental studies.…”