Tryptophan Cu(I)–π interaction fine-tunes the metal binding properties of the bacterial metallochaperone CusF

Abstract

The periplasmic metallochaperone CusF coordinates Cu(I) and Ag(I) through a unique site consisting of a Met 2 His motif as well as a Cu(I)-π interaction between a nearby tryptophan, W44, and the metal ion. Through mutational analyses we investigate here the role that W44 in CusF plays in metal coordination. Nuclear magnetic resonance spectra show that the specificity of CusF for Cu(I) and Ag(I) is not altered by mutation of W44. X-ray absorption studies reveal that W44 protects the bound Cu(I) from oxidation as well as from adventitious ligands. Competition assays demonstrate that W44 does not significantly contribute to the affinity of CusF for metal, but that substitution of W44 by methionine, which forms a fourth Cu(I) ligand, substantially increases the affinity. These studies indicate that W44 is important in maintaining a moderate-affinity and solvent-shielded threecoordinate environment for Cu(I), which has implications for the function of CusF as a metallochaperone.

Adaptor protein mediates dynamic pump assembly for bacterial metal efflux

“…CusF, although having a metal-binding affinity comparable to CusB (46,47), is not essential for CusCBA assembly, as we showed (Fig. 1C).…”

Insight into the Cation−π Interaction at the Metal Binding Site of the Copper Metallochaperone CusF

“…4,5). 9 Our calculations found that Cu + was bound to His36, Met44, Met47 and Met49 residues in a distorted tetrahedral arrangement as shown in Fig. 3.…”

“…7 Experiments by Loftin et al have suggested that Trp44 protects Cu + /Ag + from oxidative stress in the periplasm. 9 The role of the cation-π interaction in the function of CusF remains unclear as a W44M mutation exists in 25% of CusF proteins. The W44M mutant CusF is known to have a higher binding affinity for Cu + 7,9 .…”

“…While the nature of metal coordination in the cation-π interaction-lacking metal binding site of the W44M mutant has been probed with extended X-ray absorption fine structure (EXAFS), the exact geometry of the metal binding site remains to be determined. 9 …”

“…The higher interaction energy and almost two-fold stabilization energy observed in the W44M mutant are consistent with the higher binding affinity for Cu + in this mutant form. 7,9 While these calculations provide new insights about the binding environment of Cu + in W44M CusF, they are hindered by an inherent lack of conformational sampling that may have introduced errors.…”