Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase
Abstract
SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in B. subtilis. Here we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base-pair, we deduce that SpoIIIE's step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.
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© 2015, Liu et al.
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Further reading
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- Structural Biology and Molecular Biophysics
Many voltage-gated potassium (Kv) channels display a time-dependent phenomenon called C-type inactivation, whereby prolonged activation by voltage leads to the inhibition of ionic conduction, a process that involves a conformational change at the selectivity filter toward a non-conductive state. Recently, a high-resolution structure of a strongly inactivated triple-mutant channel kv1.2-kv2.1-3m revealed a novel conformation of the selectivity filter that is dilated at its outer end, distinct from the well-characterized conductive state. While the experimental structure was interpreted as the elusive non-conductive state, our molecular dynamics simulations and electrophysiological measurements show that the dilated filter of kv1.2-kv2.1-3m is conductive and, as such, cannot completely account for the inactivation of the channel observed in the structural experiments. The simulation shows that an additional conformational change, implicating isoleucine residues at position 398 along the pore lining segment S6, is required to effectively block ion conduction. The I398 residues from the four subunits act as a state-dependent hydrophobic gate located immediately beneath the selectivity filter. By mutating I398 to Asparagine, ion permeation can be resumed in the kv1.2-kv2.1-3m channel, which was not a reversion C-type inactivation, since AgTxII fails to block the ionic permeation of kv1.2-kv2.1-3m_I398N. As a critical piece of the C-type inactivation machinery, this structural feature is the potential target of a broad class of QA blockers and negatively charged activators thus opening new research directions towards the development of drugs that specifically modulate gating-states of Kv channels.
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- Structural Biology and Molecular Biophysics
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