was measured at 50 mM KCl and decreased with increasing KCl concentration. This was not due to lack of Dbp8-Esf2 association under the tested salt concentrations, as the in vitro binding of GST-Esf2 to His6-Dbp8 was not dramat- ically affected as judged by pull-down experiment with nickel-NTA beads (Figure 4B). These results suggest that Esf2 is a stimulatory factor for Dbp8 ATPase activity. To determine the mechanism of how binding of Esf2 to Dbp8 could stimulate its ATPase activity, we determined Michaelis–Menten kinetic parameters in the presence or absence of Esf2. ATP hydrolysis was measured at varying ATP concentrations (10–500 mM) and in the presence or absence of equimolar amounts of Esf2. Initial velocities (v) were calculated after 3 min of incubation at 30 C. During this time period we observed a linear correlation between Nucleic Acids Research, 2006, Vol. 34, No. 10 3195 time and the amount of ATP hydrolyzed (data not shown). Average velocities were ﬁtted to the Michaelis–Menten equation and KM (ATP) and kcat values were calculated (Figure 4C). When bound to Esf2, Dbp8 has a KM for ATP of 86 mM, 2-fold lower than the KM of Dbp8 alone (194 mM). Under the tested conditions, Dbp8 binding to Esf2 increased the kcat from 17 min 1 to 52 min 1. Thus, Esf2 stimulates Dbp8 ATP hydrolysis activity likely by increasing its afﬁnity for ATP, thereby increasing its ATPase activity. The C-terminal domain of Esf2 is required for binding to Dbp8 and for stimulating Dbp8 ATPase activity If Esf2 is indeed a co-factor for Dbp8 enzymatic activity, Esf2 binding to Dbp8 should be required for function. Bioinformatics searches for conserved protein motifs in Esf2 indicated that it had two coiled-coil domains (40–67 and 225–254 amino acids) on either side of the RRM (98– 196 amino acids). Since coiled-coil domains can function as protein–protein interaction scaffolds [reviewed in (26)], we predicted that one of these domains might be involved in Dbp8 binding. To test this, we generated a number of Esf2 deletion mutants (Figure 5A) and assayed their ability to interact with Dbp8 in a directed yeast 2-hybrid assay (Figure 5B). Deletion of the ﬁrst 90 amino acids, including the ﬁrst coiled-coil domain, (Esf2 DN) or deletion of the pre- dicted RRM motif (Figure 6A; 91–205 amino acids; Esf2 DRRM) did not noticeably impair interaction with Dbp8. In contrast, the C-terminal deletion mutant (Figure 5A; Esf2 DC) failed to interact with Dbp8 in the 2-hybrid assay (Figure 5B), suggesting that amino acids 206–317, which include one of the coiled-coil domains, are required for bind- ing to Dbp8. When tested in the ATP hydrolysis assay, the Esf2 DC mutant, which did not bind Dbp8, also no longer substantially stimulated Dbp8 activity (9-fold for Esf2 WT versus 2-fold for Esf2 DC; Figure 5C). Therefore, Esf2 Figure 4. (A) Esf2 stimulates Dbp8 ATP hydrolysis activity in vitro. A total of 10 pmol of Dbp8 was incubated with various amounts of GST-Esf2 or GST-Rpa34 (0–1.5 M excess over Dbp8, X-axis) in buffers containing 200 mM KCl, for 25 min on ice to allow complex formation to occur. ATP was then added to the reaction and ATP hydrolysis (Y-axis) was measured after 30 min of incubation at 30 C. Plotted are the averages and standard errors that were derived from three independent experiments. (B) Esf2- mediated stimulation of Dbp8 ATPase activity is optimal at lower potassium chloride concentrations. ATP hydrolysis assays were performed using 5 pmol of recombinant Dbp8 and GST-Esf2 and varying salt concentrations (50– 450 mM KCl). ATP hydrolysis was measured after 30 min of incubation at 30 C. Plotted are the averages and standard errors that were derived from three independent experiments. To determine if under these conditions GST-Esf2 and Dbp8 still form a complex, 300 pmols of each protein were incubated under the same conditions used for the ATP hydrolysis assay. Protein complexes were precipitated using Ni-NTA beads, resolved by SDS– PAGE and stained with Coomassie brilliant blue [Figure embedded in the graph shown in (B)]. (C) Dbp8 binding to Esf2 increases Dbp8 affinity for ATP and increases the ATP turnover rate. ATP hydrolysis assays were performed using 5 pmols of Dbp8 in the presence or absence of equimolar amounts of Esf2 in 50 mM KCl and various ATP concentrations. The initial velocities, plotted on the Y-axis, were determined by measuring the amount of ATP hydrolysis (mM) during a 3 min period (data not shown) in the presence of various [ATP] (10, 50, 100, 250 and 500 mM). The data were fitted to the Michaelis–Menten equation from which the KM and kcat were determined. Plotted are the averages derived from two independent experiments.