| Abstract
| - The bacterial DNA cytosine methyltransferase M.HhaI sequence-specifically modifies DNAin an S-adenosylmethionine dependent reaction. The enzyme stabilizes the target cytosine (GCGC) intoan extrahelical position, with a concomitant large movement of an active site loop involving residues80−99. We used multidimensional, transverse relaxation-optimized NMR experiments to assign nearly80% of all residues in the cofactor-bound enzyme form, providing a basis for detailed structural anddynamical characterization. We examined details of the previously unknown effects of the cofactor bindingwith M.HhaI in solution. Addition of the cofactor results in numerous structural changes throughout theprotein, including those decorating the cofactor binding site, and distal residues more than 30 Å away.The active site loop is involved in motions both on a picosecond to nanosecond time scale and on amicrosecond to millisecond time scale and is not significantly affected by cofactor binding except for afew N-terminal residues. The cofactor also affects residues near the DNA binding cleft, suggesting a rolefor the cofactor in regulating DNA interactions. The allosteric properties we observed appear to be closelyrelated to the significant amount of dynamics and dynamical changes in response to ligand binding detectedin the protein.
|
