De Novo Design of Metalloproteins and Metalloenzymes in a Three-Helix Bundle

Abstract

For more than two decades, de novo protein design has proven to be an effective methodology for modeling native proteins. De novo design involves the construction of metal-binding sites within simple and/or unrelated α-helical peptide structures. The preparation of α3D, a single polypeptide that folds into a native-like three-helix bundle structure, has significantly expanded available de novo designed scaffolds. Devoid of a metal-binding site (MBS), we incorporated a 3Cys and 3His motif in α3D to construct a heavy metal and a transition metal center, respectively. These efforts produced excellent functional models for native metalloproteins/metalloregulatory proteins and metalloenzymes. Morever, these α3D derivatives serve as a foundation for constructing redox active sites with either the same (e.g., 4Cys) or mixed (e.g., 2HisCys) ligands, a feat that could be achieved in this preassembled framework. Here, we describe the process of constructing MBSs in α3D and our expression techniques.

Keywords: De novo protein design; Metal-binding site; Metalloenzyme; Metalloprotein; Metalloregulatory protein; Protein expression; Three-helix bundle.

Fig. 1

Structures of de novo designed peptides. (a) X-ray crystal structure of As(III) bound CSL9C (PDB 2JGO), a three-stranded coiled coil scaffold. (b) Solution structure of α₃D. Apolar residues of α₃D divided into four layers, as indicated by varying shades of gray. The first layer comprises F7, L42, and L56 at the N-terminal end of the bundle. Subsequent layer contains L11, F38, and A60. The third layer has all isoleucine residues at the 14th, 35th, and 63rd positions. The C-terminal layer composes of L18, L28, and L67. These layers were predicted to provide a 3Cys metal-binding site

Fig. 2

Subsequent α₃D derivatives for heavy and transition metal binding. (a) Solution structure of α₃DIV, which exhibits a 3Cys site at positions 18, 28, and 67 that coordinates Cd, Hg, and Pb. (b) Model of a 3His α₃D derivative, α₃DH₃, which was demonstrated to bind Zn and perform the function of carbonic anhydrase. This model was constructed from the α₃DIV structure