title: Combined computational design of a zinc-binding site and a protein-protein interaction: one open zinc coordination site was not a robust hotspot for de novo ubiquitin binding.
abstract: We computationally designed a de novo protein-protein interaction between wild-type ubiquitin and a redesigned scaffold. Our strategy was to incorporate zinc at the designed interface to promote affinity and orientation specificity. A large set of monomeric scaffold surfaces were computationally engineered with three-residue zinc coordination sites, and the ubiquitin residue H68 was docked to the open coordination site to complete a tetrahedral zinc site. This single coordination bond was intended as a hotspot and polar interaction for ubiquitin binding, and surrounding residues on the scaffold were optimized primarily as hydrophobic residues using a rotamer-based sequence design protocol in Rosetta. From thousands of independent design simulations, four sequences were selected for experimental characterization. The best performing design, called Spelter, binds tightly to zinc (Kd  < 10 nM) and binds ubiquitin with a Kd of 20 µM in the presence of zinc and 68 µM in the absence of zinc. Mutagenesis studies and nuclear magnetic resonance chemical shift perturbation experiments indicate that Spelter interacts with H68 and the target surface on ubiquitin; however, H68 does not form a hotspot as intended. Instead, mutation of H68 to alanine results in tighter binding. Although a 3/1 zinc coordination arrangement at an interface cannot be ruled out as a means to improve affinity, our study led us to conclude that 2/2 coordination arrangements or multiple-zinc designs are more likely to promote high-affinity protein interactions.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Bryan S
authors.initials: BS
authors.lastname: Der
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Jha
authors.forename: Raamesh K
authors.initials: RK
authors.lastname: Jha
authors.forename: Steven M
authors.initials: SM
authors.lastname: Lewis
authors.forename: Peter M
authors.initials: PM
authors.lastname: Thompson
authors.forename: Gurkan
authors.initials: G
authors.lastname: Guntas
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D016296:Mutagenesis; D011485:Protein Binding; D060066:Protein Interaction Maps; D025801:Ubiquitin / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
chemical_list: D025801:Ubiquitin; D015032:Zinc; D000409:Alanine