Observing an induced-fit mechanism during sequence-specific DNA methylation.
The characterization of conformational changes that drive induced-fit mechanisms and their quantitative importance to enzyme specificity are essential for a full understanding of enzyme function. Here, we report on M.HhaI, a sequence-specific DNA cytosine C(5) methyltransferase that reorganizes a flexible loop (residues 80-100) upon binding cognate DNA as part of an induced-fit mechanism. To directly observe this approximately 26A conformational rearrangement and provide a basis for understanding its importance to specificity, we replaced loop residues Lys-91 and Glu-94 with tryptophans. The double mutants W41F/K91W and W41F/E94W are relatively unperturbed in kinetic and thermodynamic properties. W41F/E94W shows DNA sequence-dependent changes in fluorescence: significant changes in equilibrium and transient state fluorescence that occur when the enzyme binds cognate DNA are absent with nonspecific DNA. These real-time, solution-based results provide direct evidence that binding to cognate DNA induces loop reorganization into the closed conformer, resulting in the correct assembly of the active site. We propose that M.HhaI scans nonspecific DNA in the loop-open conformer and rearranges to the closed form once the cognate site is recognized. The fluorescence data exclude mechanisms in which loop motion precedes base flipping, and we show loop rearrangements are directly coupled to base flipping, because the sequential removal of single hydrogen bonds within the target guanosine:cytosine base pair results in corresponding changes in loop motion.
Estabrook
R August
RA
Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA.
Reich
Norbert
N
D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D018698:Glutamic Acid; D014364:Tryptophan; D004247:DNA; D008239:Lysine