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Title: A new topology of the HK97-like fold revealed in Bordetella bacteriophage by cryoEM at 3.5 Å resolution}
Article:
Abstract:
Bacteriophage BPP-1 infects and kills Bordetella species that cause whooping cough. Its diversity-generating retroelement( DGR) provides a naturally occurring phage-display system, but engineering efforts are hampered without atomic structures. Here, we report a cryo electron microscopy structure of the BPP-1 head at 3. 5 Å resolution. Our atomic model shows two of the three protein folds representing major viral lineages: jellyroll for its cement protein( CP) and HK97-like( ‘Johnson’) for its major capsid protein( MCP). Strikingly, the fold topology of MCP is permuted non-circularly from the Johnson fold topology previously seen in viral and cellular proteins. We illustrate that the new topology is likely the only feasible alternative of the old topology. β-sheet augmentation and electrostatic interactions contribute to the formation of non-covalent chainmail in BPP-1, unlike covalent inter-protein linkages of the HK97 chainmail. Despite these complex interactions, the termini of both CP and MCP are ideally positioned for DGR-based phage-display engineering.
Introduction:
Capsid proteins of non-enveloped viruses fall, so far, into three major structural classes: the β-jellyroll, the HK97 fold, and the fold of dsRNA-virus shell proteins( Bamford et al., 2005; Oksanen et al., 2012). The RNA bacteriophage MS2 subunits have a fourth structure, not yet found in eukaryotic viruses( Valegard et al., 1990). The HK97 fold is present not only in a large number of dsDNA bacteriophage capsids, including the T-phages, lambdoid phages, etc, but also in the major capsid protein of eukaryotic herpesviruses( Bamford et al., 2005). In HK97 itself, the intersubunit contacts are reinforced by a post-assembly covalent linkage—an isopeptide bond between adjacent gp5 subunits, so positioned that the entire capsid is topologically interlinked in a ‘chainmail’ arrangement( Duda, 1998; Wikoff et al., 2000). In other cases, such as bacteriophage λ, the non-covalent interactions between the subunits are reinforced by an additional ‘cement’ protein, which binds on the outer surface of the capsid at positions close to those of the isopeptide bonds in HK97( Lander et al., 2008). Heads of bacteriophage λ defective in this cement protein break down during DNA packaging( Sternberg and Weisberg, 1977; Fuller et al., 2007; Lander et al., 2008). Bacteriophage BPP-1 is a short-tailed, dsDNA virus and a member of the Podoviridae family. It infects and kills Bordetella species that cause whooping cough in humans and respiratory diseases in other mammals. It has a T = 7l icosahedral capsid, ∼670 Å in diameter. A 7 Å resolution cryoEM structure of BPP-1 showed that its capsid protein has an HK97-like fold, but the shell has an additional protein component( Dai et al., 2010). Lacking information at the time about their genetic identities, these proteins were named according to their structural roles: a major capsid protein( MCP) and a ‘cement protein’( CP) that decorates the shell. From a biotechnology standpoint, BPP-1 has emerged as an attractive phage-display system thanks to its unique and well characterized diversity-generating retro-element( DGR). As the only known source of massive DNA sequence deviation in prokaryotes, DGRs use a unique reverse transcriptase-based mechanism to introduce targeted diversity into protein-coding DNA sequences to accelerate the evolution of adaptive traits( Liu et al., 2002; Guo et al., 2008). As such, BPP-1 is a naturally occurring diversity-generating system and can be engineered to display foreign proteins with adaptive heterologous sequences. An atomic description of the BPP-1 head will enhance bioengineering efforts, reveal non-covalent molecular interactions conducive of stable bacteriophage capsid formation, and clarify evolutionary relationships of BPP-1 MCP and CP with proteins in other viruses. In this study, we report the three-dimensional( 3D) structure of the BPP-1 head at ∼3. 5 Å resolution determined by single-particle cryoEM and derived an atomic model. Both our structural and structure-based mutagenesis studies reveal major novelties in the BPP-1 MCP and CP structures and their interactions. We also show that the C-termini of both MCP and CP are ideally positioned to display DGR-diversified peptide libraries for protein engineering applications.
Discussion:
We found that BPP-1 CP and MCP adopt two of the three characteristic folds of major virus lineages, jellyroll, and HK-97/Johnson. Moreover, the topology of the Johnson fold in BPP-1 MCP is non-circularly permuted as compared to the previously known topology of the Johnson fold. Until now, the Johnson fold has been observed at atomic detail in the Siphoviridae and Myoviridae families of the three tailed-bacteriophage families, exemplified by gp5 of HK97( Siphoviridae)( Wikoff et al., 2000) and gp24 of T4( Myoviridae)( Fokine et al., 2005). Our atomic model derived from the 3. 5 Å cryoEM map of BPP-1 represents the first atomic model for a virus in the third tailed-bacteriophage family, the Podoviridae. Indeed, BPP-1 represents the first among all viruses shown to contain both the jellyroll and HK97-like folds. In the virosphere, the conventional wisdom is that viruses have evolved from a few distinctive lineages separately. Three major lineages have been proposed based on three highly distinctive structures: jellyroll-like( i. e., jellyroll lineage, include single- and double-jellyroll), HK97-like( i. e., HK97 lineage) and BTV-like( i. e., BTV lineage), and members of each lineage contain MCPs with conserved folds originating from a common ancestor( Rossmann and Johnson, 1989; Bamford et al., 2005; Abrescia et al., 2012). Our result suggests that BPP-1 is likely a hybrid virus that has adopted the characteristic structures from both the jellyroll and HK97 lineages. The head of bacteriophage Epsilon15 also contains two capsid proteins( gp7 and gp10) located at similar positions as CP( Bbp16) and MCP( Bbp17) in BPP-1. In particular, BPP-1 Bbp17( MCP) shares ∼46% sequence identity with gp7 of Epsilon15 phage, indicating that the two proteins are likely to have nearly identical structures at least at the level of protein backbones. Indeed, although initially misinterpreted to have a different topology( Figure 5—figure supplement 2H–I) due to limited resolution( Jiang et al., 2008), the model of Epsilon15 gp7 was recently updated on the basis of an improved 4. 5 Å resolution map( Baker et al., 2013) to bear the same BPP topology as we initially reported( Zhang et al., 2012) and detailed further in this study. As described in the results above, the BPP topology and HK97 topology represent the only two feasible topologies of the Johnson fold( Figure 5). To date, all other known atomic structures of proteins with the Johnson fold adopt the HK97 topology, including gp5 of HK97 in Siphoviridae( Wikoff et al., 2000), gp24 of T4 in Myoviridae( Fokine et al., 2005), a 39kD spherical particle-forming protein in archaea( Akita et al., 2007) and an encapsulin protein in bacteria( Sutter et al., 2008)( Figure 5—figure supplement 2A–E). In these two topologies, the primary positions of their β- and α-elements, are swapped in a non-circularly permutated fashion( Figure 5A–C, Figure 5—figure supplement 1), which rarely occurs in nature( Vogel and Morea, 2006). This, combined with the fact that no recognizable sequence similarity can be identified between the BPP-1 MCP and other HK97-like MCPs( CLUSTALW scores: ∼5), obscures the origin of the BPP topology. From an engineering stand point, it is fortunate that both termini of MCP and the C-terminus of CP are exposed on the external surface of the capsid, ideally positioned for peptide display. Indeed, we observed infectious phage particle formation when either CP or MCP was fused to a peptide of 22 amino acids at their C-terminus( HG and JFM, unpublished observation). As BPP-1 DGRs diversify DNA sequences in vivo, this suggests they may be engineered to display heterologous sequences on the phage capsid by fusion to the termini of CP or MCP. For DGR-based phage-display, the diversity of the DNA library is not limited by the efficiency of DNA transformation, and both the library construction and optimization can occur entirely inside bacterial cells without the need for in vitro manipulation. Moreover, the selected phage particles can be easily re-amplified for iterative selection since the tail, not the engineered head, is involved in phage-receptor recognition. The atomic model of the BPP-1 head presented in this study provides a road map forward to harness the great potentials afforded by the unique properties of BPP-1 for phage-display engineering.
Whooping cough is a respiratory illness caused by bacteria in the Bordetella genus . Among the general public , Bordetella species have become a hot topic in recent years due to the re-emergence of whooping cough in the United States and elsewhere . Scientists , meanwhile have become interested in a virus called BPP-1 that can kill the Bordetella species . BPP-1 is a double-stranded DNA virus , and such viruses have long been of interest to scientists because they are the most abundant organisms on Earth . These viruses are also noteworthy because their shells ( also known as capsids ) are capable of withstanding the very high pressures ( up to about 40 atmospheres ) that are created by packing so much DNA into the very small volume inside the capsid . BPP-1 is of particular interest because it is capable of making large-scale changes to its own DNA in order to adapt to changes in its hosts and environment . Of all the organism that do not contain nuclei within their cells ( collectively known as prokaryotes ) , BPP-1 is the only one that is capable of making such changes to its DNA . However , efforts to exploit the properties of BPP-1 for bioengineering applications have been hampered because its detailed structure is not known . Now Zhang et al . have used cryo electron microscopy to study the structure of BPP-1 at the atomic level . Most viruses belong to one of three major lineages , with each lineage having a characteristic fold in its capsid proteins . Zhang et al . found that BPP-1 contains two of these folds , which suggests that it is a hybrid of two of these lineages . This is the first time that such a structure has been observed . Moreover , Zhang et al . found that one of the folds has an unusual topology that has not been seen before . The atomic structure reveals how double-stranded DNA viruses use a variety of non-covalent interactions and a type of protein ‘chainmail’ to form a highly stable capsid that is capable of withstanding very high pressures . In addition to enabling applications in bioengineering , the new structure might also provide insights into the evolution of prokaryotes .