9MXX

Computationally Designed protein with isopeptide bond dnIPB-2

Summary for 9MXX

• Entry DOI: 10.2210/pdb9mxx/pdb
• Descriptor: De novo protein with intramolecular isopeptide bond dnIPB-2, SULFATE ION (3 entities in total)
• Functional Keywords: isopeptide, computational design, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 1
• Total formula weight: 17545.55

Authors

Srisantitham, S.,Tezcan, F.A. (deposition date: 2025-01-21, release date: 2025-04-09, Last modification date: 2025-04-23)

Primary citation

Srisantitham, S.,Walker, A.L.,Markel, U.,Tezcan, F.A.
De Novo Design of Proteins for Autocatalytic Isopeptide Bond Formation.
J.Am.Chem.Soc., 147:12338-12346, 2025

PubMed Abstract: 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.

PubMed: 40138671
DOI: 10.1021/jacs.5c03319

Experimental method

X-RAY DIFFRACTION (2.4 Å)