NMR studies of restriction enzyme-DNA interactions: role of conformation in sequence specificity

Abstract

Sequence specific DNA binding proteins are thought to adopt distinct conformations when binding to target (cognate) and nontarget (noncognate) sequences. There is both biochemical and crystallographic evidence that this behavior is important in mediating sequence recognition by the Mg(II)-dependent type II restriction enzymes. Despite this, there are few systematic comparisons of the structural behavior of these enzymes in various complexes. Here, (1)H-(15)N HSQC NMR spectroscopy is applied to PvuII endonuclease (2 x 18 kDa) in an effort to better understand the relationship between sequence recognition and enzyme conformational behavior. Spectra of the free enzyme collected in the absence and presence of metal ions indicate that while there is a modest backbone conformational response upon binding Ca(II), this does not occur with Mg(II). Substrate binding itself is accompanied by very dramatic spectral changes consistent with a large-scale conformational response. HSQC spectra of the enzyme bound to cognate (specific) and noncognate (nonspecific) oligonucleotides in the presence of Ca(II) are dramatically distinct, revealing for the first time the structural uniqueness of a PvuII cognate complex in solution. The strong correlation between NMR spectral overlap and crystallographic data (C(alpha) rmsd) permits characterization of the nonspecific PvuII complex as being more similar to the free enzyme than to the specific complex. Collectively, these data support the notion that it is the DNA, not the metal ion, which promotes a unique conformational response by the enzyme. It therefore follows that the principle role of metal ions in complex formation is one of driving substrate affinity and stability rather than conformationally priming the enzyme for substrate binding and sequence recognition. These results not only provide valuable insights into the mechanism of protein-DNA interactions but also demonstrate the utility of NMR spectroscopy in structure-function studies of these representative nucleic acid systems.