As part of intracellular copper trafficking pathways, the human copper chaperone Hah1 delivers We proposed 2-body and 3-body interaction models based on previously known structures of homologous proteins to describe both intermolecular Hah1-MBD and intramolecular MBD-MBD interactions. These interaction models and our smFRET results were then used to formulate and quantify a comprehensive Hah1-MBD34 mechanism. The enhanced interaction stability of Hah1 with the multi-MBD system, the dynamic intramolecular MBDMBD interactions, and the ability of Hah1 to interact with multiple MBDs simultaneously suggest an efficient and versatile mechanism for the Hah1-to-WDP pathway to transport Cu Last, but not least, I would like to thank my wife, Dixie, and daughter, Lilly. Their love and support was critical, and I will forever be in debt to their future vitality and happiness.

Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the cus operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.multicomponent efflux complex | substrate-responsive dynamic assembly | periplasmic adaptor protein | metal sensing | single-molecule tracking B acteria are often exposed to harsh environments, including high metal ion concentrations and toxic organic molecules. Efflux of metal ions helps bacteria maintain appropriate intracellular concentrations of essential metals while removing toxic ones (1-6). Efflux of organic molecules, including antibiotics, is a key mechanism for bacterial multidrug resistance (7-14). The tripartite resistance-nodulation-division (RND) family efflux pumps confer major clinically relevant drug resistance in Gram-negative bacteria such as Escherichia coli and the infectious Pseudomonas aeruginosa (7-14). They are composed of a proton-motive-force-driven inner-membrane pump, a periplasmic adaptor protein, and an outer-membrane channel. Once assembled, these pumps traverse the cell periplasm, providing a direct extrusion pathway from the periplasm (and cytoplasm) to the outside of the cell. However, these direct pathways also tightly link the inner and outer membranes, which, if overly stable, would impede the periplasm's plasticity and ability to respond dynamically to external and internal stimuli to buffer the cell from changes in its surroundings (15).How can tripartite efflux pumps operate without compromising the dynamic nature of the periplasm? One possibility is that these efflux complexes are dynamic structures and assemble only in the presence of their substrates. This mechanism has been hypothesized for the E. coli HlyBD-TolC complex (16), in which HlyB is a ATP-binding cassette superfamily efflux pump. However, experimental validation of this mechanism, as well as its relevance to the RND family efflux pumps, remains elusive, partly due to the difficulty in studying two membrane proteins together with a periplasmic protein under physiologically relevant conditi...