3OOR: I-SceI mutant (K86R/G100T)complexed with C/G+4 DNA substrate

Citation:
Abstract
Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G(+4) base pair for the wild-type A:T(+4) base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T(+4) were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T(+4) or the C:G(+4) base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G(+4) recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T(+4) target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G(+4) target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed approximately 36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G(+4) substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.
PDB ID: 3OORDownload
MMDB ID: 86298
PDB Deposition Date: 2010/8/31
Updated in MMDB: 2017/11
Experimental Method:
x-ray diffraction
Resolution: 2.5  Å
Source Organism:
Saccharomyces cerevisiae
Similar Structures:
Biological Unit for 3OOR: trimeric; determined by author and by software (PISA)
Molecular Components in 3OOR
Label Count Molecule
Protein (1 molecule)
1
Intron Encoded Endonuclease I-scei(Gene symbol: SCEI)
Molecule annotation
Nucleotides(2 molecules)
1
5'-d(*cp*ap*cp*gp*cp*tp*ap*gp*gp*gp*ap*tp*ap*ap*cp*cp*gp*gp*gp*tp*ap*ap*tp*ap*c)-3'
Molecule annotation
1
5'-d(*gp*gp*tp*ap*tp*tp*ap*cp*cp*cp*gp*gp*tp*tp*ap*tp*cp*cp*cp*tp*ap*gp*cp*gp*t)-3'
Molecule annotation
Chemicals (3 molecules)
1
3
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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