Intersubunit coordination in a homomeric ring ATPase

Abstract

Homomeric ring-ATPases perform many vital and varied tasks in the cell, ranging from chromosome segregation to protein degradation. Here we report the first direct observation of the inter-subunit coordination and the step size of such a ring-ATPase, the dsDNA packaging motor in the bacteriophage φ29. Using high-resolution optical tweezers, we find that packaging occurs in increments of 10 bp. Statistical analysis of the preceding dwell times reveals that multiple ATPs bind during each dwell, and application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps. These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated via a mechanism novel for ring-ATPases. In addition, a step size that is a non-integer number of base pairs demands new models for motor-DNA interactions.

Mechanistic constraints from the substrate concentration dependence of enzymatic fluctuations

“…In particular, the values of N L and N S indicate that each cycle must contain no less than two ATP binding states and no less than four additional, non-ATP-binding states. Previously, similar conclusions were drawn from the measured value of n min at the highest and lowest concentrations of ATP (5 μM and 1,000 μM ATP), under the assumption that these conditions were sufficiently limiting or saturating for ATP (29). However, limiting or saturating concentrations of [ATP] for the mean dwell time need not be limiting or saturating for n min , and, thus, it was not necessarily obvious that the values of n min measured at the lowest and highest [ATP] were accurate estimates of the asymptotic values of this parameter.…”

“…Additional measurements at high opposing loads revealed that after the dwell, the DNA was packaged not in a single step, but in a burst of four 2.5-bp steps. The observation of four steps per cycle strongly suggests that the motor loads four ATPs to four different subunits during the observed dwells (29). Previous work (29,34) has shown that the mean dwell time is well described by a Michaelis-Menten [ATP] dependence; thus, we expect that the substrate dependence of n min is fit by Eq.…”

“…From previous work, it has been established that the binding of ATP likely involves an initial, reversible docking process followed by a tight binding transition, which is irreversible and commits the nucleotide to the hydrolysis cycle (29,34). A measured value of α consistent with zero suggests that this initial docking process is not highly reversible.…”

“…This bacteriophage utilizes a pentameric ring (32) of identical ATPase subunits to drive the compaction of its dsDNA genome into a proteinacous precursor capsid during viral self-assembly (33). Recent high-resolution optical trapping measurements (29) have revealed the discrete, 10 base pair, increments of DNA packaged each cycle of the motor. The observation of the discrete steps of this motor makes it possible to compile the exact time between steps-the dwell times of the enzyme.…”

“…We discuss the kinetic parameters introduced by this expression and derive the mechanistic constraints they provide. Finally, we conclude by applying our expression to fluctuation data collected for the packaging motor of the bacteriophage φ29 (29).…”