Non-Standard Protein Engineering at the Boundary of Molecular Mechanics and Quantum Chemistry: Halogen-Based Design of Insulin Analogs

Abstract

The broad utility of halogens in medicinal chemistry has motivated application of hybrid quantum- and molecular-mechanical methods. Extending these concepts to a therapeutic protein (insulin), iodination of a conserved tyrosine at position B26 was recently shown to enhance key properties of a rapid-acting clinical analog, including its thermodynamic stability and resistance to fibrillation. Here, we (i) describe quantitative atomic-level simulations of the mono-iodininated insulin to predict its structural features and (ii) test these predictions by X-ray crystallography and multidimensional NMR spectroscopy. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog—as a dimer and hexamer—exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Eight aromatic rings at this interface (residues B16, B24-B26 and their dimer-related mates) must reorganize to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of the mono-iodonated insulin analog (determined as a zinc hexamer at 2.3 A resolution). Given that residues B24-B30 detach from the core on receptor binding, the environment of the 3-iodo-tyrosine at position B26 in a hormone-receptor complex must differ from that in the free hormone. Based on the recent structure of a “micro-receptor” complex, we predict that more marked reorientation of the modified tyrosine at the receptor interface enables directional halogen bonding and halogen-directed hydrogen bonding: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient σ-hole and electronegative equatorial band. Thus, inspired by quantum chemistry and molecular dynamics, such “halogen engineering” promises to extend principles of medicinal chemistry to proteins. Extensions of this approach to fluoro-aromatic, chloro-aromatic systems, and bromo-aromatic systems in non-standard protein design and their potential therapeutic applications will also be discussed.