The mycobacterial cell envelope acts as a multilayered barrier to drugs. However, the role of lipid composition in the properties of different mycobacterial membranes, otherwise dictating their interactions with drugs, is poorly understood. In this study, we found that hydration states, solvation relaxation kinetics, rotational lipid mobility, and lateral lipid diffusion differed between inner and outer mycobacterial membranes. Molecular modeling showed that lipid clustering patterns governed membrane dynamics in the different layers of the cell envelope. By regulating membrane properties, lipid composition and structure modulated water abundance and interactions with lipid head groups. These findings can help deepen our understanding of the physical chemistry underlying membrane structure and function, as well as the interaction of mycobacterial membranes with drugs and host membranes.

The mycobacterial cell envelope has spatially resolved inner and outer membrane layers with distinct compositions and membrane properties. However, the functional implication and relevance of this organization remain unknown. Using membrane biophysics and molecular simulations, we reveal a varied interaction profile of these layers with antibiotic Rifabutin, underlined by the structural and chemical makeup of the constituent lipids. The mycobacterial inner membrane displayed the highest partitioning of Rifabutin, which was located exclusively in the lipid head group/interfacial region. In contrast, the drug exhibited specific interaction sites in the head group/interfacial and hydrophobic acyl regions within the outer membrane. Altogether, we show that the design of membrane-active agents that selectively disrupt the mycobacterial outer membrane structure can increase drug uptake and enhance intracellular drug concentrations. Exploiting the mycobacterium-specific membrane−drug interaction profiles, chemotypes consisting of outer membrane-disruptive agents and antitubercular drugs can offer new opportunities for combinational tuberculosis (TB) therapy.

The dimerization of transactive response DNA-binding protein of 43 kDa (TDP-43) is crucial for the RNA metabolism, and the higher-order aggregation of TDP-43 would induce several neurodegenerative diseases. The dimerization and aggregation of TDP-43 are regulated by the phosphorylation on its N-terminal domain (NTD). Understanding the regulation mechanism of TDP-43 NTD dimerization is crucial for the preventing of harmful aggregation and the associated diseases. In this study, the dimerization processes of wild-type (WT), phosphorylated S48 (pS48), and phosphomimic S48E mutation (S48E) of TDP-43 NTD are characterized by the enhanced sampling technology. Our results show that the phosphorylation not only shift the conformation population of bound and unbound state of TDP-43 NTD, but also would regulate the dimerization processes, including increase the binding free-energy barrier. The phosphomimic mutation would also shift the conformational space of TDP-43 NTD dimer to the unbound structures; however, the thermodynamic and kinetic properties of the dimerization processes between the phosphorylated and phosphomimic mutant systems are distinct, which reminds us to carefully study the phosphorylation regulation by using the phosphomimic mutations.

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