9hth

Crystal structure of the fused, cysteine-less Aes123 PolB1 split intein (with S1A, N159A mutations)

spinqualitylabelsall models 9hth, resolution 1.38Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 9hth is a 2 chain structure with sequence from Aeromonas phage Aes123. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.378Å Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function I6YMS9_9CAUD Publication Abstract from PubMed 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 beta-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. A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing.,Humberg C, Yilmaz Z, Fitzian K, Dorner W, Kummel D, Mootz HD Nat Commun. 2025 Mar 19;16(1):2723. doi: 10.1038/s41467-025-57596-x. PMID:40108172 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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