8wkd

Connectase T1A C192S mutant from Methanocaldococcus mazei with peptide substrateConnectase T1A C192S mutant from Methanocaldococcus mazei with peptide substrate

Structural highlights 8wkd is a 2 chain structure with sequence from Methanosarcina mazei. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.98Å Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function A0A0F8NKN3_METMZ Publication Abstract from PubMed Enzyme-catalyzed protein modifications have become invaluable in diverse applications, outperforming chemical methods in terms of precision, conjugation efficiency, and biological compatibility. Despite significant advances in ligases, such as sortase A and OaAEP1, their use in heterogeneous biological environments remains constrained by limited target sequence specificity. In 2021, Lupas' group introduced Connectase, a family of repurposed archaeal proteases for protein ligations, but its low processivity and lack of structural information have impeded further engineering for practical biological and biophysical applications. Here, we present the X-ray crystallographic structures of MmConnectase (Methanococcus maripaludis, MmCET) in both apo and substrate-bound forms. Comparative analysis with its inactive paralogue, MjCET (Methanococcus janaschi), reveals the structural basis of MmCET's high-precision ligation activity. We propose modifications to the N-terminal substrate recognition motifs to suppress MmCET's reversible protease activity, enabling high-precision protein ligations in complex biological environments, such as serum-containing cell cultures. To further demonstrate the enhanced processivity and precision, single-molecule protein unfolding experiments showed that our optimized Connectase, in conjunction with OaAEP1(C247A), can perform stepwise tandem ligations of protein leading to a well-defined protein polymer. Structural Basis of High-Precision Protein Ligation and Its Application.,Chong KHC, Liu L, Chua R, Chai YT, Lu Z, Liu R, Tan EYJ, Dong J, Khoh YH, Lin J, Zhong FL, Lescar J, Zheng P, Wu B J Am Chem Soc. 2025 Jan 2. doi: 10.1021/jacs.4c10689. PMID:39745918 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

spinqualitylabelsall models 8wkd, resolution 1.98Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image