Abstract
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.
Highlights
Tion of real-time protein conformational rearrangements coupled to specificity have been quantitated for a small number of enzymes [17,18,19,20]
We extend our prior Substrate Preparation—DNA substrates used for kinetic and studies [29, 30] by directly probing loop motion using the fluo- fluorescence studies were all synthesized by Midlands DNA (Midrescence signal of tryptophans engineered into the flexible loop land, TX) and high-performance liquid chromatography purified
We track the reorganization of a flexible loop within M.HhaI, previously implicated in base flipping and DNA specificity
Summary
Buffer (LEB, 50 mM KH2PO4, pH 7.5, 1 M NaCl, 1 mM -mercaptoethanol, 100 M phenylmethylsulfonyl fluoride, and 20 mM imidazole). Eluted protein was tural information on cognate and nonspecific complexes sug- loaded onto a phosphocellulose column equilibrated gests that loop positioning is determined by the sequence of the with Extraction Buffer (EB, 10 mM KH2PO4, pH 7.5, 200 mM bound DNA, providing a molecular basis for an induced- NaCl, 1 mM -mercaptoethanol, and 1 mM EDTA). The temporal assignment of loop move- was washed with EB until the absorbance of the eluant at 280 ment in relation to other known steps in the reaction cycle, such nm remained unchanged over time, and the protein was eluted as base flipping, could be insightful for understanding if these in a single step with EB, which contained 800 mM NaCl. The processes are enzyme-assisted or occur passively [9, 34]. The highly conserved nature of this loop The following single strand oligonucleotide sequences were used: among DNA cytosine methyltransferases [36] suggests our results may be broadly applicable
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