N-Terminal Region of CusB Is Sufficient for Metal Binding and Metal Transfer with the Metallochaperone CusF

Abstract

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N and C termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump, CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein’s function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal, but also to play an active role in efflux.

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

“…The detailed mechanism of the CusCBAF efflux system and, in particular, the role played by the periplasmic adaptor protein CusB has been the subject of considerable debate. Two models have been proposed in the literature-the "funnel" model and the "switch" model (23,30). In the funnel model, excess periplasmic copper is sequestered by CusF, transferred to CusB (24), and then delivered to CusA for extrusion.…”

“…In the "switch" model, copper loading of CusB induces a conformational change (22), which allows CusB to bind to CusA and open the entry site on the CusA pump, which is known to be located in its periplasmic cleft (26,34). Although both models predict that either CusB deletion, or metal-site mutagenesis should lead to copper sensitive strains-an expectation that has been experimentally verified (22,23)-definitive evidence for or against either model has been lacking and is compounded by the fact that the copper-binding N-terminal domain of CusB is disordered in crystal structures of the isolated protein (27), or its complex with CusA (29). Existing evidence, although weak, has leaned more toward the switch model, based mainly on projections of the distance of the CusB metal-binding site from the CusA periplasmic cleft (30), and analogy to structural data on other metal resistance RND adaptor proteins such as ZneB of Cupriavidus metallodurans CH34.…”

“…Unlike other PAPs, which are thought to primarily provide structural support, CusB is conformationally flexible and also possesses a metal-binding site within its N-terminal region (22). Additionally, cusB-knockout strains produce a Cu-sensitive phenotype (22,23). CusF and CusB have been shown to freely exchange Ag(I) and Cu(I) toward equilibrium in highly specific protein-protein interactions (24,25).…”

“…The purpose of our study was to create such a scenario and to determine the method of activation of the CusA pump, thereby defining the exact roles of CusF and CusB in periplasmic Ag and Cu efflux. Recently, a truncated N-terminal CusB fragment (CusB NT; hereafter, CusB) that behaves similarly to native CusB in vivo has been developed, facilitating its study (23).…”

“…By observing the Fourier transform (FT) spectra derived from the Se extended X-ray absorption fine structure (EXAFS), specific holo and apo metallosites within a mixture of proteins can be clearly distinguished. In the Cus efflux system, the metal-binding ligand sets known as of yet are two Mets, one His and a weakly coordinating tryptophan for CusF (20,21,24), and three Mets each for CusB (22,23) and CusA (26), respectively. Therefore, the Cus system is an excellent candidate for Se-Met labeling, because all three of the Cus proteins under investigation contain at least two active-site Met residues.…”