Interactions between CusF and CusB Identified by NMR Spectroscopy and Chemical Cross-Linking Coupled to Mass Spectrometry

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Abstract
The Escherichia coli periplasmic proteins CusF and CusB, as part of the CusCFBA efflux system, aid in the resistance of elevated levels of copper and silver by direct metal transfer between the metallochaperone CusF and the membrane fusion protein CusB before metal extrusion from the periplasm to the extracellular space. Although previous in vitro experiments have demonstrated highly specific interactions between CusF and CusB that are crucial for metal transfer to occur, the structural details of the interaction have not been determined. Here, the interactions between CusF and CusB are mapped through nuclear magnetic resonance (NMR) spectroscopy and chemical cross-linking coupled with high-resolution mass spectrometry to better understand how recognition and metal transfer occur between these proteins. The NMR 1H−15N correlation spectra reveal that CusB interacts with the metal-binding face of CusF. In vitro chemical cross-linking with a 7.7 Å homobifunctional amine-reactive cross-linker, BS2G, was used to capture the CusF/CusB interaction site, and mass spectral data acquired on an LTQ-Orbitrap confirm the following two cross-links: CusF K31 to CusB K29 and CusF K58 to CusB K32, thus revealing that the N-terminal region of CusB interacts with the metal-binding face of CusF. The proteins transiently interact in a metal-dependent fashion, and contacts between CusF and CusB are localized to regions near their respective metal-binding sites.

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