Structural Studies of Bacteriophage α3 Assembly (original) (raw)

DNA packaging intermediates of bacteriophage φX174

Structure, 1995

Background: Like many viruses, bacteriophage X174 packages its I)NA genome into a procapsid that is assembled from structural intermediates and scaffolding proteins. The procapsid contains the structural proteins F, G and H, as well as the scaffolding proteins B and D. Provirions are formed by packaging of DNA together with the small internal J proteins, while losing at least some of the B scaffolding proteins. Eventually, loss of the I) scaffolding proteins and the remaining B proteins leads to the formation of mature virions. Results: X174 108S 'procapsids' have been purified in milligram quantities by removing 114S (mature virion) and 70S (abortive capsid) particles from crude lysates by differential precipitation with polyethylene glycol. 132S 'provirions' were purified on sucrose gradients in the presence of EDTA. Cryo-electron microscopy (cryo-EM) was used to obtain reconstructions of procapsids and provirions. Although these are very similar to each other, their structures differ greatly from that of the virion. The F and G proteins, whose atomic structures in virions were previously determined from X-ray crystallography, were fitted into the cryo-EM reconstructions. This showed that the pentamer of G proteins on each five-fold vertex changes its conformation only slightly during DNA packaging and maturation, whereas major tertiary and quaternary structural changes occur in the F protein. The procapsids and provirions were found to contain 120 copies of the I) protein arranged as tetramers on the twofold axes. IDNA might enter procapsids through one of the 30 A diameter holes on the icosahedral three-fold axes.

Conformational Switching by the Scaffolding Protein D Directs the Assembly of Bacteriophage fX174

Mol Cell, 2004

either arrested assembly or aberrant particle formation. 915 West State Street Thus, scaffolding proteins function as higher order West Lafayette, Indiana 47907 equivalents of molecular chaperones, regulating quater-2 Department of Veterinary Science and Microbiology nary structure in the same way that chaperones regulate University of Arizona tertiary structure (Dokland, 1999). Building 90 Bacteriophage systems, in which biochemical, ge-Room 201 netic, and structural data can be correlated, have been Tucson, Arizona 85721 particularly informative in the study of scaffoldingdirected assembly (Dokland et al., 1997, 1999; Fane and Prevelige, 2003; Hayashi et al., 1988; Morais et al., 2003; Summary Sun et al., 2000; Thuman-Commike et al., 2000)

Structural basis for scaffolding-mediated assembly and maturation of a ds DNA virus.

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8-and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T ¼ 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.

Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy

Nature, 2008

A half-century after the determination of the first threedimensional crystal structure of a protein 1 , more than 40,000 structures ranging from single polypeptides to large assemblies have been reported 2 . The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-Å resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (e15) particle, by singleparticle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity 3,4 . However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond e15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus φX174

Journal of Molecular Biology, 1999

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage fX174. Assembly of the fX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of à`c losed'' procapsid particle has been determined to 3.5 A Ê resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A Ê resolution electron microscopic reconstruction of thè`o pen'' procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly a-helical fold and displays remarkable conformational variability. We report here an improved and re®ned structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.

Backbone structure of the infectious e15 virus capsid revealed by electron cryomicroscopy

Nature, 2000

A half-century after the determination of the first threedimensional crystal structure of a protein 1 , more than 40,000 structures ranging from single polypeptides to large assemblies have been reported 2 . The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-Å resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (e15) particle, by singleparticle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity 3,4 . However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond e15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

Conformational Switching by the Scaffolding Protein D Directs the Assembly of Bacteriophage φX174

Molecular Cell, 2004

either arrested assembly or aberrant particle formation. 915 West State Street Thus, scaffolding proteins function as higher order West Lafayette, Indiana 47907 equivalents of molecular chaperones, regulating quater-2 Department of Veterinary Science and Microbiology nary structure in the same way that chaperones regulate University of Arizona tertiary structure (Dokland, 1999). Building 90 Bacteriophage systems, in which biochemical, ge-Room 201 netic, and structural data can be correlated, have been Tucson, Arizona 85721 particularly informative in the study of scaffoldingdirected assembly (Dokland et al., 1997, 1999; Fane and Prevelige, 2003; Hayashi et al., 1988; Morais et al., 2003; Summary Sun et al., 2000; Thuman-Commike et al., 2000)