Extended Data Fig. 5: Evolved OppA-variant OppA-iso.

a. X-ray crystal structure of OppA:G-SisoK (PDB ID: 9RD1) with mutated positions highlighted as spheres. Left: Positions mutated in error prone OppA variants 1–4. Orange spheres indicate residues that were targeted for site-saturation mutagenesis; grey spheres denote additional mutations in the error prone OppA variants. Middle: Green spheres indicate residues mutated in the final evolved variant OppA-iso. Right: Table summarizing all mutations identified in error prone OppA variants 1–4 and in OppA-iso. Residues in bold were targeted for site saturation mutagenesis. b. Structural comparison of the OppA:G-SisoK complex (PDB ID: 9RD1) with the previously published structure showing OppA bound to endogenous linear peptide ligands (PDB ID: 3TCF) with a focus on binding motifs at the C-terminal end of the bound ligands. In OppA:G-SisoK, the carboxylate of lysine is in hydrogen-bonding distance to the side chains of R439, N392 and H397. In the linear peptide bound complex (OppA:XXX), the C-terminal carboxylate of the tripeptide XXX interacts primarily with the side chain of R439 and residues N392 and H397 are not ideally positioned to contribute to strong hydrogen-bonding, suggesting that the interaction with R439 is most important for linear tripeptides. In constrast, for isopeptide-linked tripeptides such as G-SisoK the hydrogen-bond donors, H397 and N392, are well positioned to interact with the lysine carboxylate, likely stabilizing the complex also in the absence of a positively charged side chain at position 439.