Structure determination of the head–tail connector of bacteriophage ϕ29

Abstract

The head±tail connector of bacteriophage 929 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head±tail connector, surrounded by a 929-encoded, 174-base, RNA and a viral ATPase protein, both of which have ®vefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A Ê diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A Ê resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A Ê resolution using 12-fold non-crystallographic symmetry averaging and solvent¯attening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A Ê single isomorphous replacement phases were improved using density modi®cation, producing an interpretable electron-density map. The crystallographically re®ned structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2 1 2 1 2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively¯exible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Computer Modeling of Three-dimensional Structure of DNA-packaging RNA (pRNA) Monomer, Dimer, and Hexamer of Phi29 DNA Packaging Motor

“…The wide end is embedded in the capsid, and the narrow end is exposed (12,66,67). By sequence homology comparison, it was predicted that the connector protein (gp10) contains a conserved RNA recognition motif, residues 148 -214, located at the narrow end of the connector that protrudes from the procapsid (11, 31) (for a review, see Ref.…”

Diverse self-association properties within a family of phage packaging RNAs

“…1B; Bailey et al 1990;Reid et al 1994a,b;Garver and Guo 1997;Bourassa and Major 2002). The pRNA self-associates to form a ring (Simpson et al 2001;Shu et al 2007;Morais et al 2008;Ding et al 2011), which encircles the dodecameric connector protein ring using a "hand-in-hand" or "kissing loop" interaction that involves base-pairing between complementary sequences in loops of adjacent pRNA molecules ( Fig. 1C; Garver and Guo 1997;Chen et al 1999Chen et al , 2000.…”

“…The motor uses ATP hydrolysis to package ∼19.3 kb of dsDNA through its ring-like structure into the capsid in <5 min against a back pressure of ∼57 pN ( Fig. 1A; Guo et al 1987aGuo et al ,b, 2006Simpson et al 2001;Smith et al 2001;Guasch et al 2002;Serwer 2003;Guo 2005;Fuller et al 2007;Hugel et al 2007;Morais et al 2008;Rao and Feiss 2008;Aathavan et al 2009;Lee et al 2009;Fu et al 2010;Ding et al 2011). For phage phi29, the full-length transcribed pRNA is 174 nt long, but a 120 nt-long truncated version is sufficient for in vitro packaging ( Fig.…”

“…The phi29 packaging motor and phi29 packaging RNA (pRNA). (A, left) Partial model of the phi29 packaging motor based on a cryo-EM reconstruction (Morais et al 2008), the crystal structure of phi29 packaging RNA (Ding et al 2011), and the connector protein crystal structure (Simpson et al 2001). The cryo-EM density and connector protein are shown in blue and pRNA in red.…”

Efficient DNA Packaging of Bacteriophage PRD1 Requires the Unique Vertex Protein P6

“…Despite little or no sequence similarity, all portal proteins for which structures have been solved display similar folds and form multimeric ring-or cone-like structures (2,26,32,37,70,85,92). Portals that have been studied as isolated proteins have been 11-to 14-mers, but all portals analyzed within the virion have been composed of 12 subunits of the portal protein (2, 12, 22, 25, 26, 28, 30, 37, 47-49, 54, 64, 69, 70, 85, 92, 94).…”