Construction of a blue copper analogue through iterative rational protein design cycles demonstrates principles of molecular recognition in metal center formation

The construction and characterization of a series of proteins in which the Blue Copper CysHis2Met primary coordination sphere was placed in various orientations within the hydrophobic core of thioredoxin has allowed exploration of the principles of molecular recognition between proteins and metals. An automated rational protein design algorithm predicted structurally suitable locations for these centers without rise of either a potential preexisting binding site or any structural or sequence homology between the thioredoxin host and known Blue Copper proteins. A series of four primary designs and 32 variants were constructed. It was necessary to surround the designed primary coordination sphere with a hydrophobic shell to ensure the absence of potential alternative coordinating residues. Formation of a stable Cu(II)-thiolate bond required destabilization of a normally favored redox reaction in which the thiol is oxidized to a disulfide. This was achieved by more deeply burying the coordinating cysteine, presumably via a mechanism in which the free energy of protein unfolding opposes the competing redox reaction. The distorted tetrahedral coordination geometry of the Cu(II) complex is unstable with respect to a competing tetragonal geometry resulting from incorporation of bound water. Although natural systems appear to sterically exclude such water binding, this exclusion mechanism was not successfully reproduced in the designs presented here. Instead, a suitably placed small cavity allowed a strong, exogenous ligand, such as azide, to be introduced axially, which competitively stabilizes the tetrahedral geometry corresponding to a 'Type 1.5' Blue Copper complex in favor of the tetragonally bound water. This iterative rational design study demonstrates that destabilization of competing reactions ('negative design') is a crucial, if cryptic, aspect of molecular recognition in proteins, and that proteins have evolved a variety of mechanisms that impose negative design constraints.

Duke Scholars

Author Homme Wytzes Hellinga Biochemistry