6BCN

I-LtrI E184D bound to cognate substrate (pre-cleavage complex)

Summary for 6BCN

• Entry DOI: 10.2210/pdb6bcn/pdb
• Descriptor: Ribosomal protein 3/homing endonuclease-like fusion protein, DNA (26-MER), CALCIUM ION, ... (5 entities in total)
• Functional Keywords: nucleic acid, hydrolase, hydrolase-dna complex, hydrolase/dna
• Biological source: Leptographium truncatum; More
• Total number of polymer chains: 12
• Total formula weight: 209575.96

Authors

Brown, C.,Zhang, K.,McMurrough, T.A.,Gloor, G.B.,Edgell, D.R.,Junop, M. (deposition date: 2017-10-20, release date: 2018-10-24, Last modification date: 2024-03-13)

Primary citation

McMurrough, T.A.,Brown, C.M.,Zhang, K.,Hausner, G.,Junop, M.S.,Gloor, G.B.,Edgell, D.R.
Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.
Nucleic Acids Res., 46:11990-12007, 2018

PubMed Abstract: LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications.

PubMed: 30357419
DOI: 10.1093/nar/gky976

Experimental method

X-RAY DIFFRACTION (2.5 Å)