A Mets Motif Peptide Found in Copper Transport Proteins Selectively Binds Cu(I) with Methionine-Only Coordination

Abstract

Mets motifs, which refer to methionine-rich sequences found in the high-affinity copper transporter Ctr1, also appear in other proteins involved in copper trafficking and homeostasis, including other Ctrs as well as Pco and Cop proteins isolated from copper-resistant bacteria. To understand the coordination chemistry utilized by these proteins, we studied the copper binding properties of a peptide labeled Mets7-PcoC with the sequence Met-Thr-Gly-Met-Lys-Gly-Met-Ser. By comparing this sequence to a series of mutants containing noncoordinating norleucine in place of methionine, we confirm that all three methionine residues are involved in a thioether-only binding site that is selective for Cu(I). Two independent methods, one based on mass spectrometry and one based on rate differences for the copper-catalyzed oxidation of ascorbic acid, provide an effective K(D) of approximately 2.5 microM at pH 4.5 for the 1:1 complex of Mets7-PcoC with Cu(I). These results establish that a relatively simple peptide containing an MX(2)MX(2)M motif is sufficient to bind Cu(I) with an affinity that corresponds well with its proposed biological function of extracellular copper acquisition.

Bioinorganic chemistry of synucleinopathies: Deciphering the binding features of Met motifs and His-50 in AS–Cu(I) interactions

“…Our findings are consistent with previous work indicating that the affinity of Met-rich sites for Cu(I) depends not only of the number of Met residues but also the number of amino acids separating them [27][28][29][30]. Indeed, in model peptides containing two Met residues the affinity N HSQC spectra of AS in the absence and presence of increasing Cu(I) concentrations, from red to green (darker to lighter gray in the print version): 0 to 5 equivalents of Cu(I).…”

“…All experiments were recorded on protein samples dissolved in buffer MES 20 mM, NaCl 100 mM, pH 6.5 at 15°C. was shown to range from 15-20 μM to 45-50 μM when the number of intervening amino acids between both Met residues changed from two to five, respectively [28]. Thus, a more drastic effect, likely the one observed in this work, might be expected for the affinity of Cu(I) to the 116 MPVDPDNEAYEM 127 fragment.…”

Crucial role of copper in detection of metal-coordinating odorants

“…Future crystallization studies may elucidate the structure of the copper-MTMT complex. Our proposed model of copper simultaneously binding to OR protein residues and thiol/ thiolate as well as thioether or selenoether groups in odorants MeX(CH 2 ) n SH may be analogous to the binding that occurs in blue copper proteins, which involves simultaneous binding to cysteine and methionine (or selenomethionine) (39)(40)(41). Sophisticated molecular dynamics simulation studies may be required to resolve the precise mode of interaction among the receptor-ligand-metal trio associated with odorant docking.…”

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

“…As mentioned above, the bound Cu(I) ion was found to coordinate residues M573, M623 and M672. These three methionines specifically form a typical threemethionine coordination site for binding Cu(I)/Ag(I) [43,44]. Binding of Cu(I) initiates significant conformational changes in the periplasmic as well as transmembrane domains of CusA.…”