Monitor protein complex assembly using surface plasmon resonance
What is it about?
MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB–TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA–MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex.
Why is it important?
In Escherichia coli, MacB is the only ABC-type exporter implicated in resistance against antibiotics in this bacterium. The homologues of MacB were identified in all bacteria with some of them implicated in secretion of toxins and siderophores. So far only few labs in the world are doing research on this protein complex and only 15 papers has been published related to this topic. My research is especially valuable because I developed a highly sensitive real-time assay in monitoring the tripartite complex assembly in the presence of hydrolysable ATP, which could be used in the studies of protein-protein or protein- small molecule interactions of all kinds. I also demonstrated the role of ATP in MacB dimerization and MacAB complex assembly, which provided a new insight on understanding how the ATP-driven MDR efflux systems function efficiently. I was able to reconstitute MacB into lipid nanodiscs and retain the ATP hydrolysis activity of MacB in lipid environment. Most importantly I identified a critical segment of MacB, the periplasmic loop, which serves as an essential component for the interactions of MacB with MacA and TolC. This loop could be considered as a potential drug target and lead to the discovery of new drugs, which aim on interrupting the complex formation of MacAB-TolC. Overall, my research helps to understand the transport mechanism of MacAB-TolC system. This will provide us more information to the MDR phenomenon in bacteria and probably serve as a prototype for the study of MacAB homologs. Furthermore, no study of MDR in lipid nanodiscs has been reported. Therefore, my research on reconstitution of MacAB sets the first example and could benefit the study of MDR in lipid environment.
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