Biophysical Insights into Daptomycin’s Mechanisms: Solid-state NMR Characterisation of Lipids and Proteins

Abstract

The rapid emergence of multi- and pan-drug–resistant bacteria has made antimicrobial resistance (AMR) a critical global health crisis, as traditional antibiotics lose effectiveness and new drug development stalls. Lipid-targeting antibiotics offer a promising alternative by disrupting bacterial membranes—an action less prone to resistance—but their supramolecular mechanisms (e.g., oligomerization on membrane surfaces) complicate detailed study. To address this, the thesis employs solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy alongside complementary biophysical methods to reveal, at atomic resolution, how lipid-targeting antibiotics interact with bacterial membranes under near-physiological conditions. ssNMR excels at characterizing biomolecules in native, heterogeneous environments, providing unique insights into molecular interactions, dynamics, and structural diversity. By integrating ssNMR with techniques such as isothermal titration calorimetry (ITC), this work maps binding interfaces, monitors conformational changes, and captures dynamic behavior of antibiotics within lipid bilayers—advancing our understanding in both microbiology and neurodegeneration. The thesis is organized into three parts. Part I focuses on isolating and characterizing isotopically labeled bacterial lipids. Part II investigates the mode of action of daptomycin, a key lipid-targeting antibiotic, using ssNMR and ITC. Part III applies ssNMR to study mutant huntingtin exon 1 fibrils linked to Huntington’s disease. Part I: Lipid Isolation and Characterization We purified uniformly 13C-labeled diglucosyldiacylglycerol (Glc2-DAG) from Staphylococcus simulans and incorporated it into complex membrane mimetics. ssNMR data reveal Glc2-DAG’s organization within bilayers and underscore its role as the lipoteichoic acid (LTA) anchor. Given its impact on membrane properties and daptomycin resistance, these findings highlight the value of advanced NMR in decoding lipid contributions to antibiotic susceptibility. Part II: Daptomycin Mode of Action Daptomycin remains a last-line defense against multidrug-resistant Gram-positive pathogens, including MRSA and VRE. In Chapter 3, ssNMR and ITC characterize daptomycin’s calcium-bound trimeric form binding with high affinity to the anionic headgroup of phosphatidylglycerol (PG). Key contacts involve the phosphate and chiral centers of the PG headgroup, and associated structural and dynamic changes are mapped in both lipid and peptide. We also assess interactions with cardiolipin and lysyl-PG, revealing how lipid composition influences antibiotic potency and resistance. Chapter 4 details the biosynthesis and purification of 13C,15N-labeled daptomycin from Streptomyces roseosporus, enabling high-resolution 1H-detected ssNMR studies of the daptomycin–PG–calcium complex in membranes. We observe significant conformational rearrangements upon lipid binding and find no evidence for direct interaction with Lipid II, challenging prior models. Together, these chapters provide a refined, membrane-centric view of daptomycin’s mechanism and suggest strategies for designing next-generation lipid-targeting antibiotics. Part III: Huntington’s Disease Fibril Structure Chapter 5 uses ssNMR to dissect the architecture of polyQ15 and Q44 huntingtin exon 1 amyloid fibrils. An antiparallel β-sheet core coexists with a semi-rigid surface exhibiting distinct side-chain dynamics. The N-terminal N17 segment displays mixed α-helical and disordered conformations, while the proline-rich C-terminal domain shows a gradient of flexibility. These insights into fibril heterogeneity and surface dynamics lay a molecular foundation for targeting neurodegenerative aggregates. Conclusion This thesis demonstrates how ssNMR, combined with biophysical methods, can unravel the molecular basis of lipid-targeting antibiotics and protein aggregates under biologically relevant conditions. By delivering atomic-level descriptions of antibiotic–lipid interactions and fibril structures, it paves the way for rational design of new therapeutics against antibiotic resistance and neurodegeneration.