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
Guest exchange in an M4L6 supramolecular host has been evaluated to determine whether host rupture is required for guest ingress and egress. Two mechanistic models were evaluated: one requiring partial dissociation of the host structure to create a portal for guest passage and one necessitating deformation of the host structure to create a dilated aperture for guest passage without host rupture. Three related lines of inquiry support the nondissociative guest exchange mechanism. (a) Equally facile guest exchange is observed in labile ([Ga4L6]12-) and inert ([Ti4L6]8- and [Ge4L6]8-) hosts. (b) Molecular mechanics calculations demonstrate that the structural deformations required for enlargement of an M4L6 aperture in a nonrupture or nondissociative guest exchange mechanism are plausible. (c) As predicted by the calculations, CoCp*2+, a sterically demanding guest, significantly inhibits guest exchange. These results bring new insight to the application of the M4L6 supramolecular host for encapsulated reaction chemistry for which there are now several examples.
Over the last decade, cysteine thiolate ligands have been shown to be critical to the Cu(I) (cuprous) binding chemistry of many cytosolic metallochaperone and metalloregulatory proteins involved in copper physiology. More recently, the thioether group of methionine has begun to emerge as an important Cu(I) ligand for trafficking proteins in more oxidizing cellular environments.
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