Computational Design of an Unnatural Amino Acid Dependent Metalloprotein with Atomic Level Accuracy

Genetically
encoded unnatural amino acids could facilitate the
design of proteins and enzymes of novel function, but correctly specifying
sites of incorporation and the identities and orientations of surrounding
residues represents a formidable challenge. Computational design methods
have been used to identify optimal locations for functional sites
in proteins and design the surrounding residues but have not incorporated
unnatural amino acids in this process. We extended the Rosetta design
methodology to design metalloproteins in which the amino acid (2,2′-bipyridin-5yl)­alanine
(Bpy-Ala) is a primary ligand of a bound metal ion. Following initial
results that indicated the importance of buttressing the Bpy-Ala amino
acid, we designed a buried metal binding site with octahedral coordination
geometry consisting of Bpy-Ala, two protein-based metal ligands, and
two metal-bound water molecules. Experimental characterization revealed
a Bpy-Ala-mediated metalloprotein with the ability to bind divalent
cations including Co2+, Zn2+, Fe2+, and Ni2+, with a Kd for
Zn2+ of ∼40 pM. X-ray crystal structures of the
designed protein bound to Co2+ and Ni2+ have
RMSDs to the design model of 0.9 and 1.0 Å respectively over
all atoms in the binding site.