Solution NMR Structures of IgG Binding Domains with Artificially Evolved High Levels of Sequence Identity but Different Folds^†,‡

We describe here the solution NMR structures of two IgG binding domains with highly homologous sequences but different three-dimensional structures. The proteins, G311 and A219, are derived from the IgG binding domains of their wild-type counterparts, protein G and protein A, respectively. Through a series of site-directed mutations and phage display selections, the sequences of G311 and A219 were designed to converge to a point of high-level sequence identity while keeping their respective wild-type tertiary folds. Structures of both artificially evolved sequences were determined by NMR spectroscopy. The main chain fold of G311 can be superimposed on the wild-type α/β protein G structure with a backbone rmsd of 1.4 Å, and the A219 structure can be overlaid on the wild-type three-α-helix protein A fold also with a backbone rmsd of 1.4 Å. The structure of G311, in particular, accommodates a large number of mutational changes without undergoing a change in the overall fold of the main chain. The structural differences are maintained despite a high level (59%) of sequence identity. These proteins serve as starting points for further experiments that will probe basic concepts of protein folding and conformational switching.