9HTH
Crystal structure of the fused, cysteine-less Aes123 PolB1 split intein (with S1A, N159A mutations)
This is a non-PDB format compatible entry.
Summary for 9HTH
| Entry DOI | 10.2210/pdb9hth/pdb |
| Descriptor | DNA-directed DNA polymerase (2 entities in total) |
| Functional Keywords | intein, protein splicing, cysteine-less, ligation, splicing |
| Biological source | Aeromonas phage Aes123 More |
| Total number of polymer chains | 2 |
| Total formula weight | 39587.50 |
| Authors | Fitzian, K.,Yilmaz, Z.,Humberg, C.,Mootz, H.D.,Kuemmel, D. (deposition date: 2024-12-19, release date: 2025-05-07) |
| Primary citation | Humberg, C.,Yilmaz, Z.,Fitzian, K.,Dorner, W.,Kummel, D.,Mootz, H.D. A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing. Nat Commun, 16:2723-2723, 2025 Cited by PubMed Abstract: Split inteins catalyze protein trans-splicing by ligating their extein sequences while undergoing self-excision, enabling diverse protein modification applications. However, many purified split intein precursors exhibit partial or no splicing activity for unknown reasons. The Aes123 PolB1 intein, a representative of the rare cysteine-less split inteins, is of particular interest due to its resistance to oxidative conditions and orthogonality to thiol chemistries. In this work, we identify β-sheet-dominated aggregation of its N-terminal intein fragment as the origin of its low (~30%) splicing efficiency. Using computational, biochemical, and biophysical analyses, we characterize the fully active monomeric fraction and pinpoint aggregation-prone regions. Supported by a crystal structure, we design stably monomeric mutants with nearly complete splicing activity. The optimized CLm intein (Cysteine-Less and monomeric) retains the wild-type's ultra-fast reaction rate and serves as an efficient, thiol-independent protein modification tool. We find that other benchmark split inteins show similar precursor aggregation, suggesting that this general phenomenon arises from the intrinsic challenge to maintain the precursor in a partially disordered state while promoting stable folding upon fragment association. PubMed: 40108172DOI: 10.1038/s41467-025-57596-x PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.378 Å) |
Structure validation
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