De NovoDesign of Proteins for Autocatalytic IsopeptideBond Formation

Isopeptide bonds (IPBs)formed between the amine group of a Lys residue and the carboxamide/carboxy group of Asn/Gln or Asp/Gluplay essential roles in many biological processes, ranging from cellular signaling and regulation to blood clotting and bacterial pathogenesis. The formation of IPBs is not a spontaneous process and requires enzymatic machinery that provides a specialized active site environment to enable this challenging catalytic reaction. Here we report the de novo design and characterization of two proteins (dnIPB-1 and dnIPB-2) capable of autocatalytic IPB formation. While these designed proteins preserve the key active-site residues of their structural template (the bacterial pilin protein RrgA), they possess less than 31% sequence identity to RrgA. Extensive structural and Ala-scanning analyses indicate that IPB formation requires a solvent-protected core motif composed of several critical residues, yet there is also a large tolerance to different protein topologies and overall protein sizes in terms of accommodating an IPB-forming motif. Notably, the structural insights gained from the study of dnIPB-1 and dnIPB-2 also guided the redesign of an initially failed construct (dnIPB-3) and enabled it to form an IPB, highlighting the value of de novo design in examining sequence–structure–function relationships not explored in natural evolution. Our study highlights the versatility of IPBs as designable elements which can be used to construct functional proteins or protein-based materials with enhanced chemical, thermal, and mechanical stabilities.