Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gramnegative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn 2+ alone. The crystal structure of ZneB to 2.8 Å resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the β-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.Cupriavidus metallidurans CH34 | heavy-metal resistance | resistance nodulation cell division | periplasmic adaptor protein M icroorganisms depend on protective mechanisms to survive the effects of toxic compounds in the environment. In Gramnegative bacteria, resistance nodulation cell division (RND) -driven efflux systems confer resistance to many drugs and heavy metals (1, 2). The canonical RND-based system is formed by the association of three components: one integral to the plasma membrane, one integral to the outer membrane, and a periplasmic connector. The plasma membrane protein (RND) is a substrate/ proton antiporter. The outer-membrane factor (OMF) spans a large part of the periplasm and provides the exit portal. The periplasmic membrane fusion protein (MFP) links these two components together. Based on the nature of their substrate, tripartite RND-driven efflux systems are divided into two subclasses: the hydrophobe/amphiphile efflux (HAE) and the heavy-metal efflux (HME) subclasses. The selectivity and function of many HAE-RND systems such as the Acr and Mex families have been determined, alongside crystal structures of the RND, OMF, and MFP components of various systems (3-13). However, knowledge of HME-RND systems, including substrate specificity and the basis for selectivity, is much more limited.The integral plasma membrane components of the RND-based efflux systems are thought of as the pump; however, the discovery of increasing functions of MFPs shows that these proteins also play a major role in substrate transport. Several MFPs bind their respective substrates (14-16), facilitate substrate transport (17, 18), and are essential for transport in vitro (17). MFPs are also involved in OMF recruitment (19), and are also found in Grampositive bacteria, where no OMF is pr...

Efflux pumps belonging to the ubiquitous resistance-nodulationcell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two Xray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.T he resistance-nodulation-cell division (RND) superfamily, named based on the original members' roles in metal resistance, root nodulation, and cell division (1), is found in all kingdoms of life and is comprised of nine phylogenetically distinct families (2-5). Functional characterization of RND proteins has shown that they are transmembrane efflux pumps that transport a variety of substrates out of cells, powered by an electrochemical proton gradient (6). In Gram-negative bacteria, RND pumps are specific for toxic substrates and largely belong to one of two families; the heavy metal efflux (HME) family and the multidrug hydrophobe/amphiphile efflux-1 (HAE1) family. For the HME-and HAE1-RND-driven efflux systems, a trimer of the RND pump in the plasma membrane is coupled by a hexamer of a periplasmic membrane fusion protein (MFP), also specific for the metal ion substrate in HME-RND-driven efflux systems, to a pore formed by a trimeric outer membrane factor (OMF), forming a continuous conduit that spans the inner and outer membranes (7-10). Each protomer of the RND trimer consists of a transmembrane domain of 12 transmembrane α-helices and two large hydrophilic loops that comprise the substratebinding porter (or pore) domain and the OMF-coupling docking domain (11).X-ray crystal structures of only four RND efflux pumps have been described, including just one of the HME family to date, shedding some light on the conformational changes that are necessary for transport (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In two of these structures, those of the HAE1 efflux pumps AcrB from Escherichia coli and of the closely related MexB from Pseudomonas aeruginosa, each protomer of the homotrimer is trapped in a unique conformation, with only a single protomer conducive for substrate binding, suggestive of a functionally rotating mechanism (12, ...

Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.

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Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site.