Mycobacterial phosphatidylinositol mannosides (PIMs) and metabolically derived cell wall lipoglycans play important roles in host-pathogen interactions, but their biosynthetic pathways are poorly understood. Here we focus on Mycobacterium smegmatis PimA, an essential enzyme responsible for the initial mannosylation of phosphatidylinositol. The structure of PimA in complex with GDP-mannose shows the two-domain organization and the catalytic machinery typical of GT-B glycosyltransferases. PimA is an amphitrophic enzyme that binds mono-disperse phosphatidylinositol, but its transferase activity is stimulated by high concentrations of non-substrate anionic surfactants, indicating that the early stages of PIM biosynthesis involve lipid-water interfacial catalysis. Based on structural, calorimetric, and mutagenesis studies, we propose a model wherein PimA attaches to the membrane through its N-terminal domain, and this association leads to enzyme activation. Our results reveal a novel mode of phosphatidylinositol recognition and provide a template for the development of potential antimycobacterial compounds.

Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.

Malonyl‐CoA is an essential intermediate in fatty acid synthesis in all living cells. Here we demonstrate a new role for this molecule as a global regulator of lipid homeostasis in Gram‐positive bacteria. Using in vitro transcription and binding studies, we demonstrate that malonyl‐CoA is a direct and specific inducer of Bacillus subtilis FapR, a conserved transcriptional repressor that regulates the expression of several genes involved in bacterial fatty acid and phospholipid synthesis. The crystal structure of the effector‐binding domain of FapR reveals a homodimeric protein with a thioesterase‐like ‘hot‐dog’ fold. Binding of malonyl‐CoA promotes a disorder‐to‐order transition, which transforms an open ligand‐binding groove into a long tunnel occupied by the effector molecule in the complex. This ligand‐induced modification propagates to the helix‐turn‐helix motifs, impairing their productive association for DNA binding. Structure‐based mutations that disrupt the FapR–malonyl‐CoA interaction prevent DNA‐binding regulation and result in a lethal phenotype in B. subtilis, suggesting this homeostatic signaling pathway as a promising target for novel chemotherapeutic agents against Gram‐positive pathogens.