Molecular basis for the activation of Pseudomonas aeruginosa MsbA by Zn2+
Proteins involved in the biogenesis of lipopolysaccharide (LPS), a lipid exclusive to Gram-negative bacteria, are promising candidates for drug discovery. Specifically, the ABC transporter MsbA plays a crucial role in translocating an LPS precursor from the cytoplasmic to periplasmic facing leaflet of the inner membrane, and small molecules that inhibit its function exhibit bactericidal activity. Here, we use native mass spectrometry (MS) to determine lipid binding affinities of MsbA from P. aeruginosa (PaMsbA), a Gram-negative bacteria associated with hospital-acquired infections, in different conformations. We show the ATPase activity of the transporter is stimulated by Zn2+ and successfully trapping the protein with vanadate requires Zn2+ not Mg2+, which is necessary to trap MsbA from E. coli. We also present cryogenic-electron microscopy structures of PaMsbA in occluded and open outward-facing conformations determined to a resolution of 2.98 and 2.72 angstroms, respectively. The structures reveal a triad of histidine residues and mutation of these residues abolishes Zn2+ stimulation of PaMsbA activity. Together our studies provide detailed insight into PaMsbA structure, lipid binding preferences, and uncover a mechanism through which Zn2+ promotes the dimerization of the transporter, resulting in enhanced ATPase activity.