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

Abstract

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 crystallographic analysis of the designed protein shows only slight deviation from the computationally designed model.

Computational design of a homotrimeric metalloprotein with a trisbipyridyl core

“…A previous study in which Bpy-ala was used to engineer a novel metal-binding site in a protein scaffold highlighted important considerations for protein design efforts using this NCAA (6). In a design in which the Bpy-ala residue was not well constrained by packing interactions, we observed the formation of a highly stable [Fe(Bpy-ala f ) 2 (Bpy-ala p )]…”

“…Because Bpy forms very stable octahedral complexes with a number of biologically relevant divalent cations (e.g., Fe ), an appropriately placed Bpy-ala residue could potentially be used to generate self-assembling proteins nucleated by [M(Bpy) 3 ] 2+ complexes (where M is a divalent cation that forms an octahedral complex with Bpy). Although we (6) and others (7)(8)(9) have engineered proteins in which Bpy-ala served in structural or functional capacities, to our knowledge, the possibility of using this NCAA to drive or stabilize the formation of a protein complex has not been explored.…”

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

“…Despite the successful identification of a suitable pair of incorporation site and non-natural amino acid, the approach was based on a very qualitative visualization technique and cannot easily be applied to other systems [33]. Recently, RosettaDesign approach was successfully utilized to design a metalloprotein variant containing a non-natural amino acid, (2,2 -bipyridin-5yl) alanine, for the enhanced binding toward various metal ions [63]. In order to develop a strategy more applicable to other systems, we utilized the computational analysis tools (RosettaLigand and RosettaDesign) to evaluate various incorporation sites.…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…3A) (Mills et al 2013). Using the software Rosetta, Mills et al (2013) generated a BpyAla-containing metalloprotein with high affinity (K d for Zn 2þ is 37 pM) against a number of divalent cations. The three-dimensional structure determined by X-ray crystallography was very close to that of the design model (Fig.…”