NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

Abstract

<jats:title>Summary</jats:title><jats:p>Double-stranded DNA viruses use ATP-powered molecular motors to package their genomes. To do so, these motors must efficiently transition between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes dimerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination.</jats:p>

Atomistic Basis of Force Generation, Translocation, and Coordination in a Viral Genome Packaging Motor

“…S1 ) 18 . Like φ29 gp16, the N-terminal domain adopts the canonical ASCE ATPase fold, while the C-terminal domain is a “vestigial nuclease domain” 11 . The linker domain (residues 225 to 260) adopts a helix-loop-helix fold, again like φ29 gp16 and reminiscent of the lid subdomain identified in other ASCE ATPases such as AAA+ and helicases 1 .…”

“…The monomer in isolation (right) is color coded to match the motif classification in panel A, and is shown in two side views, from the exterior of the motor (top) and the interior of the lumen (bottom). canonical ASCE ATPase fold, while the C-terminal domain is a "vestigial nuclease domain" similar to φ29 C-terminal domain (Mahler et al, 2020). The linker domain (residues 225 to 260) adopts a helixloop-helix fold, reminiscent of the lid subdomain identified in other ASCE ATPases such as AAA+ and helicases (Erzberger and Berger, 2006).…”

“…In addition to being well suited for SMSF studies, the relatively small size and simplicity of φ29 has facilitated advanced genetic, biochemical, and structural studies (Morais, 2012). All components of the φ29 packaging system have been thoroughly characterized, and a robust highly efficient in vitro DNA packaging system has been developed (Grimes et al, 2002).Furthermore, atomic resolution structures of all individual φ29 motor components are available (Ding et al, 2011;Mahler et al, 2020;Mao et al, 2016a;Simpson et al, 2000) and medium resolution structures of motors assembled on capsids in various stages of assembly and/or packaging have been determined (Koti et al, 2008;Mao et al, 2016b;Morais et al, 2005Morais et al, , 2008Simpson et al, 2000;Woodson et al, 2020). These results indicate that the DNA packaging motor consists of a dodecameric portal protein, a pentameric prohead RNA (pRNA), and a pentameric ATPase (gene product 16; gp16) that assemble as co-axial rings at a unique vertex of the φ29 capsid.…”

Biochemical and Biophysical Characterization of the dsDNA packaging motor from theLactococcus lactisbacteriophage asccphi28

“…Since φ29 packages a unit length genome, its packaging motor is accordingly simpler. There is no need for a nuclease function to cut the DNA, and thus the φ29 ATPase lacks a fully functional nuclease domain and is only ~ 60% the size of ATPases/large terminases in genome-cutting packaging systems [16]. Similarly, the portal protein is only ~60% of the size of portals in cutting systems [17], possibly because it does not have or need any structural components to sense that the capsid is full and/or transmit this signal to the rest of the motor.…”

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

“…Since φ29 packages a unit-length genome, its packaging motor is accordingly simpler. There is no need for a nuclease function to cut the DNA, and thus the φ29 ATPase lacks a fully functional nuclease domain and is only ~ 60% the size of ATPases/large terminases in genome-cutting packaging systems [16]. Similarly, the portal protein is only ~60% of the size of the portals in the cutting systems [17], possibly because it does not have or need any structural components to sense that the capsid is full and/or transmit this signal to the rest of the motor.…”