DbpA transiently samples the closed state in the absence of ATP. (A) PRE experiments were performed with spin labeled HP92 RNA and ILMVA-labeled DbpA in the absence of ADP/BeF3. The RNA construct is shown on the left. The position of the 4-thiouridine residue that carries the nitroxide spin label is indicated by a green circle. The methyl groups of DbpA are colored according to the decrease in signal intensity, due to the spatial proximity of the spin label from blue (no effect) to red (strong reduction). The closed conformation (left structure) and an arbitrary open conformation (right structure) are depicted. The pink arrow indicates the reorientation of the RecA_N domain between the two structures. The C1′ atom of the spin labeled 4-thiouridine is shown as a green sphere. (B) PRE experiments with an hp-HP92 RNA containing a spin label in the loop of the substrate hairpin (green circle; left). Methyl groups are colored as in (A) for the closed conformation (left structure) and for an arbitrary open conformation (right structure). The pink arrows indicate the reorientation of the RecA_N domain. The methyl groups of M114 and M161 that exhibit the largest PREs in the RecA_N domain are labeled. The RNA is shown in the conformation observed in the DbpA/hp-HP92 complex, where the substrate interacts with the active site of the helicase core. (C) DbpA in the closed conformation colored according to its electrostatic surface potential (blue = positive, red = negative). (D) PRE experiments with identical RNA as in (B), but in the presence of ADP/BeF3. Methyl groups are colored as in (A) and DbpA is shown in the closed conformation.
