Revealing Solvent-Dependent Folding Behavior of Mycolic Acids from Mycobacterium Tuberculosis by Advanced Simulation Analysis

<p>Mycobacterium tuberculosis remains a persistent pathogen, partly due to its lipid rich cell wall, of which mycolic acids (MAs) are a major component. The fluidity and conformational flexibilities of different MAs in the bacterial cell wall significantly influence its properties, function, and observed pathogenicity, thus a proper conformational description of different MAs in different environments (e.g. in vacuum, in solution, in monolayers) can inform about their potential role in the complex setup of the bacterial cell wall. Previously, we have shown that molecular-dynamics (MD) simulations of MA folding <i>in vacuo</i>can be used to characterise MA conformers in seven groupings relating to bending at the functional groups (W, U and Z-conformations). Providing a new OPLS-based forcefield parameterisation for the critical cyclopropyl group of MAs and extensive simulations in explicit solvents (TIP4P water, hexane) we now present a more complete picture of MA folding properties together with improved simulation analysis techniques. We show that the ‘WUZ’ distance-based analysis can be used pinpoint conformers with hairpin bends at the functional groups, with these conformers constituting only a fraction of accessible conformations. Applying principle component analysis (PCA) and refinement using free energy landscapes (FELs), we are able to discriminate a complete and unique set of conformational preferences for representative alpha-, methoxy-, and keto-MAs, with overall preference for folded conformations. A control backbone-MA without any mero-chain functional groups showed significantly less folding in the mero-chain, confirming the role of functionalisation in directing folding. Keto-MA showed the highest percentage of WUZ-type conformations and, in particular, a tendency to fold at its alpha-methyl trans-cyclopropane group, in agreement with results from Villeneuve <i>et al.</i>MAs demonstrate similar folding in vacuum and water, with a majority of folded conformations around the W-conformation, although the molecules are more flexible in vacuum than in water. Exchange between conformations, with a disperse distribution that includes unfolded conformers, is common in hexane for all MAs, although with more organisation for Keto-MA. Globular, folded conformations are newly defined and may be specifically relevant in biofilms.</p>