Cu(I) recognition via cation-π and methionine interactions in CusF

Abstract

Methionine-rich motifs have an important role in copper trafficking factors, including the CusF protein. Here we show that CusF uses a new metal recognition site wherein Cu(I) is tetragonally displaced from a Met 2 His ligand plane toward a conserved tryptophan. Spectroscopic studies demonstrate that both thioether ligation and strong cation-π interactions with tryptophan stabilize metal binding. This novel active site chemistry affords mechanisms for control of adventitious metal redox and substitution chemistry.In recent years, metal-specific gene regulatory and cation-trafficking proteins have been isolated and demonstrate metal binding motifs with unprecedented coordination chemistry tailored to their function 1 . For example, the CXXC sequence, found in cytosolic copper chaperones and trafficking proteins, provides for facile Cu(I) transfer via low-coordinationnumber anionic intermediates 1,2 . Extracellular or periplasmic copper trafficking domains, however, function in environments that are more oxidizing than the cytosol and frequently have less well understood methionine-rich sequences 3-8 . The cus operon encodes a bacterial copper homeostasis system with several methionine-motif proteins 5,9,10 , including the periplasmic protein CusF, which is thought to serve as copper chaperone or regulator 5,6 . CusF binds Cu(I) in vitro 11 , and a methionine-rich Cu(I) site was proposed 6 based on an apo-CusF structure and NMR chemical shift data. Here we show that metal recognition in CusF involves a strong interaction between a cationic Cu(I)-thioether/imidazole center and the aromatic ring of tryptophan. To our knowledge, such cation-π interactions have not been reported for transition metal receptors or metalloenzyme active sites.Correspondence should be addressed to T.V.O. (t-ohalloran@northwestern.edu). 6 These authors contributed equally to this work.Published online at http://www.nature.com/naturechemicalbiology Reprints and permissions information is available online at

Transcriptional and Functional Studies of Acidithiobacillus ferrooxidans Genes Related to Survival in the Presence of Copper

“…Recent crystal structures of E. coli CusF revealed an intriguing Cu-binding site, HXXXXXX XWXXMXMXF (15), that includes tryptophan. The close proximity of this amino acid to Cu suggested an unusual cation-interaction between Cu(I) and the aromatic ring of tryptophan (37). Figure 1 shows the presence in A. ferrooxidans of an ORF coding for a protein with 25% identity to CusF from E. coli but with a different genomic organization since it is located distantly from cusCBA Af and divergently from copA2 Af .…”

Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex

“…These methionine pairs are M410 of TM4 and M501 of TM6; M403 of TM4 and M486 of TM6; and M391 of TM4 and M1009 of TM12 (figure 3a). It is known that copper tolerance proteins, such as CusF [27,28], CueR [45] and Atx1 [46], frequently use two-methionine or twocysteine binding pockets to carry their Cu(I)/Ag(I) cargos. Thus, these methionine pairs could potentially form binding sites for Ag(I) and Cu(I) in the transmembrane region of the pump.…”

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

“…CusCFBA is composed of the inner membrane-bound proton-substrate antiporter CusA, the periplasmic adapter protein (PAP) CusB, and the outer membrane factor (OMF) CusC (17,18). The Cus system is distinguished from other RND-type efflux pumps such as AcrB-TolC and its homologs (19), both by its association with the periplasmic metallochaperone CusF (20,21) and by the active involvement of its periplasmic adapter protein. 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).…”

“…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.…”