d ‐Cysteine Ligands Control Metal Geometries within De Novo Designed Three‐Stranded Coiled Coils

Although metal ion binding to naturally occurring l ‐amino acid proteins is well documented, understanding the impact of the opposite chirality ( d ‐)amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a d ‐configuration cysteine within a designed l ‐amino acid three‐stranded coiled coil in order to enforce a precise coordination number on a metal center. The d chirality does not alter the native fold, but the side‐chain re‐orientation modifies the sterics of the metal binding pocket. l ‐Cys side chains within the coiled‐coil structure have previously been shown to rotate substantially from their preferred positions in the apo structure to create a binding site for a tetra‐coordinate metal ion. However, here we show by X‐ray crystallography that d ‐Cys side chains are preorganized within a suitable geometry to bind such a ligand. This is confirmed by comparison of the structure of Zn II Cl(CSL16 D C) 3 2− to the published structure of Zn II (H 2 O)(GRAND‐CSL12AL16 L C) 3 − .[1][L. Ruckthong, 2016] Moreover, spectroscopic analysis indicates that the Cd II geometry observed by using l ‐Cys ligands (a mixture of three‐ and four‐coordinate Cd II ) is altered to a single four‐coordinate species when d ‐Cys is present. This work opens a new avenue for the control of the metal site environment in man‐made proteins, by simply altering the binding ligand with its mirror‐imaged d configuration. Thus, the use of non‐coded amino acids in the coordination sphere of a metal promises to be a powerful tool for controlling the properties of future metalloproteins.