Membrane proteins, accounting for 25 - 75% of the membrane mass depending on the cell type, play key roles in a wide range of physiological processes. One of the specialized functions is to transport various substrates across the membrane, part of which is facilitated by a large protein superfamily - ATP-binding cassette (ABC) transporters. In this study, we employed native mass spectrometry (MS) as well as structural strategies to characterize two ABC transporters: ABCB10 and MsbA, found in human and bacteria, respectively. ABCB10 resides in the inner membrane of mitochondria and is implicated in erythropoiesis. It is revealed here that ABCB10 binds avidly to cardiolipin with significantly higher affinity than other phospholipids using native MS. The first three binding events of cardiolipin display positive cooperativity, suggestive of specific cardiolipin binding sites on ABCB10. Functional assays show cardiolipin regulates the ATPase activity of ABCB10 in a dose-dependent fashion. Taken together, ABCB10 has a high binding affinity for cardiolipin, the high abundance of which is the characteristic of mitochondria, and this lipid also regulates the ATPase activity of the transporter. Meanwhile, MsbA plays a pivotal role in lipopolysaccharide (LPS) biogenesis in bacteria. This study demonstrates that the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2- lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over adenosine 5'-diphosphate (ADP). Guided by this observation, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 angstrom-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle. The study went further to MsbA from Pseudomonas aeruginosa, one of the ESKAPE pathogens. 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. In essence, our studies contribute to the understanding of the interactions with ligands, function, structure and mechanism of membrane proteins, with implications for drug discovery.