A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

Abstract

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, by direct counting using single-molecule fluorescence, we have determined that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive subunit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

Insights into a viral motor: the structure of the HK97 packaging termination assembly

“…Translocation of DNA against mounting internal pressure requires ATPase activity, provided by of a ring of five large terminase subunits (2)(3)(4)(5)(6). Each monomer constitutes an N-terminal ATPase (NTD) (28) and C-terminal endonuclease domain (CTD) adjoined by a linker.…”

“…A cryo-EM reconstruction of the intact Φ29 packaging motor, comprising the capsid, pRNA, large terminase and DNA, revealed the five ATPase domains, in a "cracked" helical conformation, stabilised by ATP𝛾S (3) This conformation contrasts planar structures seen for the apo Φ29 ATPase (33,34) and the ADP-bound form of highly related phage ascc-φ28 large terminase (40). The transition between the cracked helical and planar states likely represents the burst phase, which has inspired a unique DNA translocation model (3,40). However, other terminase-packaging systems may well vary wildly from the Φ29 model, which unusually utilises pRNA and does not require small terminase proteins (41,42).…”