Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease

摘要

Promiscuous mutant EcoRl endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRl*) sites more rapidly than does wild-type. The crystal structure of the A1 38T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr1 38 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleoticle while excluding two bound water molecules seen in the wildtype complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A1 38T protein to the GAATTC site has Delta S degrees more favorable and Delta H degrees less favorable than for wild-type enclonuclease binding. The interactions of Thr1 38 with flanking bases may permit A1 38T, unlike wild-type enzyme, to form complexes with EcoRl* sites that structurally resemble the specific wildtype complex with GAATTC.