Observing an induced fit mechanism during sequence specific DNA methylation

Abstract

Induced fit mechanisms rely on conformational changes that have been evoked by enzymes to facilitate a variety of processes which assist in catalysis. When the DNA cytosine C5 methyltransferase M. HhaI binds cognate DNA, a loop of 20 amino acids (80–100) makes a large 26 Å movement into the minor groove of the DNA with concomitant extrusion of the target cytosine, or baseflipping. The loop is seen in the open conformation in the AdoHcy/non-cognate DNA structure and in the closed conformation in the AdoHcy/cognate DNA structure. To observe this conformational switch by fluorescence we engineered novel tryptophans into the loop to replace K91 and E94. To better observe the differential quenching between loop conformers for K91W and E94W, the lone native tryptophan in M. HhaI (W41F) was removed. The double mutant W41F/E94W has minimal differences in kinetic and thermodynamic properties from WT. We observed a large change in W41F/E94W fluorescence by steady state and stopped-flow upon cognate DNA addition which was not observed with non-specific DNA or cofactor additions. This suggests the enzyme binds tightly to DNA at the cognate site by repositioning the loop. We also show loop motion is coupled to baseflipping and specificity. Our solution based results provide a mechanism whereby the induced-fit process related to sequence recognition is coupled to a conformational transition within the enzyme. Funding provided by the UC GREAT grant.