The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

Abstract

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a gamma-phosphate attack mechanism. ADP-P(i) and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P(i) to bind rRNA with >10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P(i) release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P(i)-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.

(Eq. 1)

Figure 1. Steady state ATPase activity of DbpA

A. PTC-RNA concentration-dependence of DbpA steady state turnover velocity at saturating (2 mM) ATP. B. ATP concentration-dependence of DbpA steady-state turnover velocity at saturating (120 nM) PTC-RNA. The solid lines through the data points are the best fits to Eq. 9. Uncertainty bars represent the standard errors from the fits. The steady-state ATPase rate is presented as the number of ATP molecules hydrolyzed per DbpA per second.

Figure 2. Kinetics of mantATP binding to DbpA and DbpA·PTC-RNA

A. Time courses of mantATP binding assayed by resonance energy transfer from DbpA tryptophans to bound mantATP. The smooth lines through the data represent the best fits to double exponentials. Final concentrations: 1 μM DbpA-PTC-RNA and (lower to upper) 30 μM, 40 μM, 60 μM and 100 μM mantATP. B. Time course of 50 μM mantATP binding to 1 μM DbpA (green) or 1 μM DbpA·PTC-RNA (blue). The solid lines are the best fits to single (DbpA) or double (DbpA-PTC) exponentials. C. [mantATP]-dependence of the fast observed rate constant of binding to DbpA () or DbpA·PTC (). Solid lines are the fits to Eq. 33 of Appendix.. D. [mantATP]-dependence of the slow observed rate constant for binding to DbpA·PTC-RNA (). The solid line represents the best fit to Eq. 34 of Supplementary Material with the apparent K_d constrained to 51 μM as predicted from the ratio of the rate constants determined from data in Panel C. Uncertainty bars represent standard errors in the fits.

Figure 3. Kinetics of mantADP binding to DbpA-PTC-RNA

A. Time courses of mantADP binding assayed by resonance energy transfer from DbpA tryptophans to bound mantADP. The smooth lines through the data represent the best fits to double exponentials. Final concentrations: 1 μM DbpA·PTC-RNA and (lower to upper) 10 μM, 20 μM, 30 μM, 40 μM and 60 μM mantADP. B. [mantATP]-dependence of the observed rate constant binding to DbpA and DbpA·PTC (fast phase () and slow phase (). Both solid lines are the fits to Eq. 2. C. The [mantADP]-dependence of the slow observed rate constant of binding mantADP to DbpA-PTC-RNA. Uncertainty bars represent standard errors in the fits. The solid line is the best fit to Eq. 2. D. Time course of mantADP dissociation from DbpA-PTC-RNA after mixing an equilibrated sample of 1 μM DbpA-PTC-RNA and 125 μM mantADP with 2 mM MgADP. The smooth lines are the best fits of the data to double exponentials with a fast observed rate constant of 169.8 (± 4.5) s^-1 comprising 82% of the amplitude and a slow observed rate constant of 39.7 (± 2.7) s^-1.

Figure 4. Transient kinetics of P_i release from DbpA-PTC-RNA

A. Time courses of transient P_i release after mixing 1 μM DbpA-PTC-RNA with excess ATP in the presence of 5 μM (final) MDCC-P_iBiP. Solid lines are the best fits to Eq. 38 in Appendix. B. [ATP]-dependence of the observed rate constant that describe the lag phase component of the time courses. The solid line is the fit using Eq. 6.

Figure 5. Distribution of ¹⁸O-labeled P_i species after hydrolysis of ATP by DbpA-PTC-RNA in 49% ¹⁸O-water

Inset-Distribution of ¹⁸O-2 and ¹⁸O-3 species. Note that only a trace of ¹⁸O-3 species due to natural abundance will be detected if there was no exchange.

Figure 6. Distribution of the DbpA-RNA biochemical states populated during steady-state ATP cycling

HR represents DbpA- RNA complex, HRT represents DbpA- RNA-ATP, HRDP_i represents DbpA-RNA-ADP-P_i, and HRD^T represents the sum of both DbpA-RNA-ADP states depicted in Scheme 1.

Scheme 1. The rRNA-activated DbpA ATPase cycle

The predominant cycling pathway at saturating rRNA is highlighted in green. The intermediates populated in the absence of RNA are colored red.