An integrative structural model of the full-length gp16 ATPase in bacteriophage phi29 DNA packaging motor

Abstract

Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. Most of these biomotors belong to a group of enzymes known as ATPases, which adopt a multi-subunit ring-shaped structure and hydrolyze adenosine triphosphate (ATP) to generate forces. The gene product 16 (gp16), an ATPase in bacteriophage {square}29, is among the most powerful biomotors known. It can overcome substantial resisting forces from entropic, electrostatic, and DNA bending sources to package double-stranded DNA (dsDNA) into a preformed protein shell (procapsid). Despite numerous studies of the {square}29 packaging mechanism, a structure of the full-length gp16 is still lacking, let alone that of the packaging motor complex that includes two additional molecular components: a connector gp10 protein and a prohead RNA (pRNA). Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment of gp16-CTD with related RNase H-like nuclease domains revealed a nucleic acid binding surface in gp16-CTD, whereas no nuclease activity has been detected for gp16. Subsequent molecular dynamics (MD) simulations showed that this nucleic acid binding surface is likely essential for pRNA binding. Furthermore, our simulations of a full-length gp16 structural model highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid, providing structural support to the previously proposed inchworm model. Lastly, we assembled an atomic structural model of the complete {square}29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage {square}29 and suggested how the individual domains of gp16 work together to power such translocation. ABSTRACT (SHORT)Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. The gene product 16 (gp16) in bacteriophage {square}29 is among the most powerful biomotors known, which adopts a multi-subunit ring-shaped structure and hydrolyzes ATP to package double-stranded DNA (dsDNA) into a preformed procapsid. Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment and molecular dynamics (MD) simulations revealed an essential binding surface of gp16-CTD for prohead RNA (pRNA), a unique component of the motor complex. Furthermore, our simulations highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid. Lastly, we assembled an atomic structural model of the complete {square}29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage {square}29 and suggested how the individual domains of gp16 work together to power such translocation.