Designed Metal‐Mediated Protein Dimerization

Abstract

The specificity and stability of protein/protein interactions is of vital interest to the pharmaceutical and biotechnological industries. Incorporation of metal‐binding sites at target protein interfaces may be one approach to improve the specificity and affinity of naturally occurring and designed protein‐protein interactions. The main goal for this research was to generate a metal‐mediated protein interface and study the effects of interfacial modification on protein‐protein affinity. Our approach recently resulted in the creation of a high‐affinity, zinc‐mediated protein homodimer.Using a metal‐templated approach we engineered novel metal binding sites to generate a high affinity interaction using the β1 domain of Streptoccocal Protein G (Gβ1). Metal coordination enables focused surface redesign, allowing for the preservation of naturally occurring or designed interfaces. Incorporation of metal binding sites generates concerted interactions allowing for enhanced affinity and specificity for protein interactions. Our protein complex is driven by metal coordination through histidine side‐chains, allowing for a metal‐dependent interaction.Complex formation and assembly was evaluating using size exclusion chromatography and x‐ray crystallography. To accurately measure the molecular weight of the protein variants we have used size‐exclusion chromatography with multi‐angle light scattering (SEC‐MALS). This analytical technique first separates proteins based on size, and the molecular weight is determined from scattered laser light. The Gβ1 monomer and metal‐mediated dimers had molecular weight of 6.2 and 12.2–13.1 kDa, respectively. Crystal structure of the dimeric proteins proved the target design was achieved and that zinc was bound at the designed interface.We have also engineered a library of rationally designed proteins that contain mutations in proximity to the metal binding sites. The goal is to exploit and explore key thermodynamic parameters that are essential for protein complex formation. We measured the binding affinity of the metal‐mediated protein‐protein interactions using a fluorescence polarization competition assay. The measured affinities were stronger than 100 nM, which is both physiologically relevant and provides additional support for the design of high‐affinity metal‐mediated protein‐protein interactions.Support or Funding InformationThis work was supported by the Department of Defense and by the California Metabolic Research Foundation.Brian is a recipient of an Arne N. Wick Pre‐doctoral Research Fellowship from the California Metabolic Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.