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Publication Abstract from PubMed

Isopeptide bonds (IPBs) horizontal line formed between the amine group of a Lys residue and the carboxamide/carboxy group of Asn/Gln or Asp/Glu horizontal line 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.

De Novo Design of Proteins for Autocatalytic Isopeptide Bond Formation.,Srisantitham S, Walker AL, Markel U, Tezcan FA J Am Chem Soc. 2025 Mar 26. doi: 10.1021/jacs.5c03319. PMID:40138671^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.