MOLECULAR REPLACEMENT USING A 50% HOMOLOGOUS NMR TRIAL MODEL

Caitriona Dennis¹ and Richard Pauptit²

¹Dept of Biochemistry & Molecular Biology, Leeds University, Leeds, LS2 9JT, UK. ²Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield SK10 4TG, UK.
Email: richard.pauptit@alderley.zeneca.com

Keywords: Molecular Replacement, NMR, colicin, immunity protein, toxins.

Molecular replacement using NMR trial models has always been difficult due to the lack of similarity between the trial model and target structure. There are only few examples of success, and these tend to be limited to cases where there is 100% sequence homology between the trail model and the target protein, i.e., cases where one is determining a crystal structure that has been previously determined by NMR. However, success is far from guaranteed even with 100% homology.

We have been struggling with a molecular replacement and refinement problem where the homology is only 50%, and were finally successful, not so much due to our own ingenuity, but rather due to currently available molecular replacement and maximum likelihood refinement software. Thus, we believe that current crystallographic technology has reached the stage where considerably increased use can be made of NMR trial models for structure solution.

The structure solved was that of a colicin immunity protein, Im7, which binds and inhibits the DNase toxin colicin E7 thereby preventing the producing cell committing suicide. The complex is specific, extremely tight, and is secreted from E.coli. The NMR structure of a 50% homologous immunity protein, Im9, which binds colicin E9, was used to solve the structure. The rotation function solution ranked 57^th, and the program refmac was instrumental to successful refinement. Details of structure solution and implications for use of NMR trial models in molecular replacement will be discussed.