The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry.

Electron microscopy in combination with image processing is a powerful method for obtaining structural information on non-crystallized biological macromolecules at the 10-50 A resolution level. The processing of noisy microscopical images requires advanced data processing methodologies in which one must carefully avoid the introduction of any form of bias into the data set. Using a novel multivariate statistical approach to the analysis of symmetry, we studied the structure of the bacteriophage SPP1 portal protein oligomer. This portal structure, ubiquitous in icosahedral bacteriophages which package dsDNA, is located at the site of symmetry mismatch between a 5-fold vertex of the icosahedral shell and the 6-fold symmetric (helical) tail. From previous studies such 'head-to-tail connector' structures were generally accepted to be homododecamers assembled in a 12-fold symmetric ring around a central channel. Using a new analysis methodology we have found that the phage SPP1 portal structure exhibits 13-fold cyclical symmetry: a new point group organization for oligomeric proteins. A model for the DNA packaging mechanism by 13-fold symmetric portal protein assemblies is presented which attributes a coherent functional meaning to their unusual symmetry.     

Preliminary crystallographic analysis of the bacteriophage P22 portal protein

Portal proteins are components of large oligomeric dsDNA pumps connecting the icosahedral capsid of tailed bacteriophages to the tail. Prior to the tail attachment, dsDNA is actively pumped through a central cavity formed by the subunits. We have studied the portal protein of bacteriophage P22, which is the largest connector characterized among the tailed bacteriophages. The molecular weight of the monomer is 82.7 kDa, and it spontaneously assembles into an oligomeric structure of approximately 1.0 MDa. Here we present a preliminary biochemical and crystallographic characterization of this large macromolecular complex. The main difficulties related to the crystallization of P22 portal protein lay in the intrinsic dynamic nature of the portal oligomer. Recombinant connectors assembled from portal monomers expressed in Escherichia coli form rings of different stoichiometry in solution, which cannot be separated on the basis of their size. To overcome this intrinsic heterogeneity we devised a biochemical purification that separates different ring populations on the basis of their charge. Small ordered crystals were grown from drops containing a high concentration of the kosmotropic agent tert-butanol and used for data collection. A preliminary crystallographic analysis to 7.0-A resolution revealed that the P22 portal protein crystallized in space group I4 with unit cell dimensions a=b=409.4A, c=260.4A. This unit cell contains a total of eight connectors. Analysis of the noncrystallographic symmetry by the self-rotation function unambiguously confirmed that bacteriophage P22 portal protein is a dodecamer with a periodicity of 30 degrees. The cryo-EM reconstruction of the dodecahedral bacteriophage T3 portal protein will be used as a model to initiate phase extension and structure determination.     

Probing conserved helical modules of portal complexes by mass spectrometry-based hydrogen/deuterium exchange

The Double-stranded DNA bacteriophage P22 has a ring-shaped dodecameric complex composed of the 84 kDa portal protein subunit that forms the central channel of the phage DNA packaging motor. The overall morphology of the P22 portal complex is similar to that of the portal complexes of Phi29, SPP1, T3, T7 phages and herpes simplex virus. Secondary structure prediction of P22 portal protein and its threading onto the crystal structure of the Phi29 portal complexes suggested that the P22 portal protein complex shares conserved helical modules that were found in the dodecameric interfaces of the Phi29 portal complex. To identify the amino acids involved in intersubunit contacts in the P22 portal ring complexes and validate the threading model, we performed comparative hydrogen/deuterium exchange analysis of monomeric and in vitro assembled portal proteins of P22 and the dodecameric Phi29 portal. Hydrogen/deuterium exchange experiments provided evidence of intersubunit interactions in the P22 portal complex similar to those in the Phi29 portal that map to the regions predicted to be conserved helical modules.     

Determination of stoichiometry and conformational changes in the first step of the P22 tail assembly

Large oligomeric portal assemblies have a central role in the life-cycles of bacteriophages and herpesviruses. The stoichiometry of in vitro assembled portal proteins has been a subject of debate for several years. The intrinsic polymorphic oligomerization of ectopically expressed portal proteins makes it possible to form rings of diverse stoichiometry (e.g., 11-mer, 12-mer, 13-mer, etc.) in solution. In this study, we have investigated the stoichiometry of the in vitro-assembled portal protein of bacteriophage P22 and characterized its association with the tail factor gp4. Using native mass spectrometry, we show for the first time that the reconstituted portal protein (assembled in vitro using a modified purification and assembly protocol) is exclusively dodecameric. Under the conditions used here, 12 copies of tail factor gp4 bind to the portal ring, in a cooperative fashion, to form a 12:12 complex of 1.050 MDa. We applied tandem mass spectrometry to the complete assembly and found an unusual dimeric dissociation pattern of gp4, suggesting a dimeric sub-organization of gp4 when assembled with the portal ring. Furthermore, native and ion mobility mass spectrometry reveal a major conformational change in the portal upon binding of gp4. We propose that the gp4-induced conformational change in the portal ring initiates a cascade of events assisting in the stabilization of newly filled P22 particles, which marks the end of phage morphogenesis.     

Structural organisation of the head-to-tail interface of a bacterial virus

In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-na?ve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.     

Two different oligomeric states of the RuvB branch migration motor protein as revealed by electron microscopy

In prokaryotes, the RuvA, B, and C proteins play major roles at the late stage of DNA homologous recombination, where RuvB complexed with RuvA acts as an ATP-dependent motor for branch migration. The oligomeric structures of negatively stained and frozen hydrated RuvB from Thermus thermophilus HB8 were investigated by electron microscopy. RuvB oligomers free of DNA formed a ring structure of about 14 nm in diameter. The averaged top view image clearly indicated a sevenfold symmetry, suggesting that it exists as a heptamer. The RuvB oligomers complexed with duplex DNA formed a smaller ring of about 13 nm in diameter. The averaged top view images represented a sixfold symmetry. This difference in oligomerization indicates that the oligomeric structure of RuvB may convert from a heptamer to a hexamer upon DNA binding. In addition, this finding provides the lesson that great care should be taken in investigating the subunit organizations of DNA binding proteins, because their oligomeric states are more sensitive to DNA interactions than expected.     

Z-Phe-Ala-diazomethylketone (PADK) disrupts and remodels early oligomer states of the Alzheimer disease Aβ42 protein

The oligomerization of the amyloid-β protein (Aβ) is an important event in Alzheimer disease (AD) pathology. Developing small molecules that disrupt formation of early oligomeric states of Aβ and thereby reduce the effective amount of toxic oligomers is a promising therapeutic strategy for AD. Here, mass spectrometry and ion mobility spectrometry were used to investigate the effects of a small molecule, Z-Phe-Ala-diazomethylketone (PADK), on the Aβ42 form of the protein. The mass spectrum of a mixture of PADK and Aβ42 clearly shows that PADK binds directly to Aβ42 monomers and small oligomers. Ion mobility results indicate that PADK not only inhibits the formation of Aβ42 dodecamers, but also removes preformed Aβ42 dodecamers from the solution. Electron microscopy images show that PADK inhibits Aβ42 fibril formation in the solution. These results are consistent with a previous study that found that PADK has protective effects in an AD transgenic mouse model. The study of PADK and Aβ42 provides an example of small molecule therapeutic development for AD and other amyloid diseases.     

The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid

During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 +/- 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 +/- 1.1 and 5.0 +/- 0.7 nm, respectively, and a height of 19.5 +/- 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as phi29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.     

Intrinsic versus imposed curvature in cyclical oligomers: the portal protein of bacteriophage SPP1.

Large cyclical oligomers may be formed by (curvi-) linear polymerization of monomers until the n(th) monomer locks in with the first member of the chain. The subunits in incomplete structures exhibit a natural curvature with respect to each other which can be perturbed when the oligomer closes cyclically. Using cryo-electron microscopy and multivariate statistical image processing we report herein a direct structural observation of this effect. A sub-population (approximately 15%) of incomplete oligomers was found within a sample of SPP1 bacteriophage portal proteins embedded in vitreous ice. Whereas the curvature between adjacent subunits of the closed circular 13-fold symmetric oligomer is 27.7 degrees, in these incomplete oligomers the angle is only 25.8 degrees, a value which almost allows for a 14-subunit cyclical arrangement. A simple model for the association of large cyclical oligomers is suggested by our data.     

Structure–Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.     

Structural transformations accompanying the assembly of bacteriophage P22 portal protein rings in vitro

The Salmonella typhimurium bacteriophage P22 assembles an icosahedral capsid precursor called a procapsid. The oligomeric portal protein ring, located at one vertex, comprises the conduit for DNA entry and exit. In conjunction with the DNA packaging enzymes, the portal ring is an integral component of a nanoscale machine that pumps DNA into the phage head. Although the portal vertex is assembled with high fidelity, the mechanism by which a single portal complex is incorporated during procapsid assembly remains unknown. The assembly of bacteriophage P22 portal rings has been characterized in vitro using a recombinant, His-tagged protein. Although the portal protein remained primarily unassembled within the cell, once purified, the highly soluble monomer assembled into rings at room temperature at high concentrations with a half time of approximately 1 h. Circular dichroic analysis of the monomers and rings indicated that the protein gained alpha-helicity upon polymerization. Thermal denaturation studies suggested that the rings contained an ordered domain that was not present in the unassembled monomer. A combination of 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS) binding fluorescence studies and limited proteolysis revealed that the N-terminal portion of the unassembled subunit is meta-stable and is susceptible to structural perturbation by bis-ANS. In conjunction with previously obtained data on the behavior of the P22 portal protein, we propose an assembly model for P22 portal rings that involves a meta-stable monomeric subunit.     

The polydispersity of αB-crystallin is rationalized by an interconverting polyhedral architecture

We report structural models for the most abundant oligomers populated by the polydisperse molecular chaperone αB-crystallin. Subunit connectivity is determined by using restraints obtained from nuclear magnetic resonance spectroscopy and mass spectrometry measurements, enabling the construction of various oligomeric models. These candidate structures are filtered according to their correspondence with ion-mobility spectrometry data and cross-validated by using electron microscopy. The ensuing best-fit structures reveal the polyhedral architecture of αB-crystallin oligomers, and provide a rationale for their polydispersity and facile interconversion.     

Atomic models by cryo-EM and site-directed spin labeling: application to the N-terminal region of Hsp16.5

We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5 has been previously studied by both cryo-EM and X-ray crystallography. The crystal structure revealed a roughly spherical protein shell with dodecameric symmetry; however, residues 1-32 were found to be disordered. The cryo-EM reconstruction at 13 A resolution appeared similar to the crystal structure but with additional internal density corresponding to the N-terminal regions of the 24 subunits. In this study, a systematic application of site-directed spin labeling and EPR spectroscopy was carried out. By combining the EPR constraints from spin label accessibilities and proximities with the cryo-EM density, we obtained an atomic model for a portion of the Hsp16.5 N-terminal region in the context of the oligomeric complex.     

In vitro packaging of DNA of the Bacillus subtilis bacteriophage SPP1

In vitro packaging of bacteriophage SPP1 DNA into procapsids is described and the requirements of this process were determined. Combination of proheads with an extract supplying terminase, DNA and phage tails yielded up to 10(7 )viable phages per milliliter of in vitro reaction under optimized conditions. The presence of neutral polymers and polyamines had a concentration and type dependent effect in the packaging reaction. The terminase donor extract lost rapidly activity at 30 degrees C in contrast to the stability of the prohead donor extract. Maturation to infective virions was observed using both procapsids assembled in SPP1 infected cells and procapsid-like structures assembled in Escherichia coli that overexpressed the SPP1 prohead gene clusters. Neither a majority of aberrant capsid-related structures present in the latter material nor procapsids lacking the portal protein inhibited DNA packaging. Addition of purified portal protein reduced DNA packaging activity in vitro only at concentrations 20-fold higher than those found in the SPP1 infected cell. The SPP1 DNA packaged in vitro originated exclusively from the terminase donor extract. This packaging selectivity was not observed in vivo during mixed infections. The data are compatible with a model for processive headful DNA packaging in which terminase and DNA co-produced in the same cell are tightly associated and can effectively discriminate the portal vertex of DNA packaging-proficient proheads from aberrant structures, from portal-less procapsids, and from isolated portal protein.     

Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids. Predominant formation of T=7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure.     

Cryo-EM elucidates mechanism of action of bacterial pore-forming toxins

Pore-forming toxins (PFTs) rupture plasma membranes and kill target cells. PFTs are secreted as soluble monomers that undergo drastic structural rearrangements upon interacting with the target membrane and generate transmembrane oligomeric pores. A detailed understanding of the molecular mechanisms of the pore-formation process remains unclear due to limited structural insights regarding the transmembrane oligomeric pore states of the PFTs. However, recent advances in the field of cryo-electron microscopy (cryo-EM) have led to the high-resolution structure determination of the oligomeric pore forms of diverse PFTs. Here, we discuss the pore-forming mechanisms of various PFTs, specifically the mechanistic details contributed by the cryo-EM-based structural studies.     

The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory

The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically assembled subunits. The c15-mer reported here is the largest c ring that is isolated and does not show the classical c-ring mismatch to the three-fold symmetry of the F1 domain.     

Portal fusion protein constraints on function in DNA packaging of bacteriophage T4

Architecturally conserved viral portal dodecamers are central to capsid assembly and DNA packaging. To examine bacteriophage T4 portal functions, we constructed, expressed and assembled portal gene 20 fusion proteins. C-terminally fused (gp20-GFP, gp20-HOC) and N-terminally fused (GFP-gp20 and HOC-gp20) portal fusion proteins assembled in vivo into active phage. Phage assembled C-terminal fusion proteins were inaccessible to trypsin whereas assembled N-terminal fusions were accessible to trypsin, consistent with locations inside and outside the capsid respectively. Both N- and C-terminal fusions required coassembly into portals with approximately 50% wild-type (WT) or near WT-sized 20am truncated portal proteins to yield active phage. Trypsin digestion of HOC-gp20 portal fusion phage showed comparable protection of the HOC and gp20 portions of the proteolysed HOC-gp20 fusion, suggesting both proteins occupy protected capsid positions, at both the portal and the proximal HOC capsid-binding sites. The external portal location of the HOC portion of the HOC-gp20 fusion phage was confirmed by anti-HOC immuno-gold labelling studies that showed a gold 'necklace' around the phage capsid portal. Analysis of HOC-gp20-containing proheads showed increased HOC protein protection from trypsin degradation only after prohead expansion, indicating incorporation of HOC-gp20 portal fusion protein to protective proximal HOC-binding sites following this maturation. These proheads also showed no DNA packaging defect in vitro as compared with WT. Retention of function of phage and prohead portals with bulky internal (C-terminal) and external (N-terminal) fusion protein extensions, particularly of apparently capsid tethered portals, challenges the portal rotation requirement of some hypothetical DNA packaging mechanisms.     

Cryo-electron Microscopy Reveals the Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC) is a giant protein assembly that penetrates the double layers of the nuclear membrane. The overall structure of the NPC has approximately eightfold symmetry and is formed by approximately 30 nucleoporins. The great size and complexity of the NPC have hindered the study of its structure for many years until recent breakthroughs were achieved by integrating the latest high-resolution cryo-electron microscopy (cryo-EM), the emerging artificial intelligence-based modeling and all other available structural information from crystallography and mass spectrometry. Here, we review our latest knowledge of the NPC architecture and the history of its structural study from in vitro to in situ with progressively improved resolutions by cryo-EM, with a particular focus on the latest subnanometer-resolution structural studies. The future directions for structural studies of NPCs are also discussed.     

Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery

The three-dimensional structure of the bacteriophage T7 head-to-tail connector has been obtained at 8A resolution using cryo-electron microscopy and single-particle analysis from purified recombinant connectors. The general morphology of the T7 connector is that of a 12-folded toroidal homopolymer with a channel that runs along the longitudinal axis of the particle. The structure of the T7 connector reveals many structural similarities with the connectors from other bacteriophages. Docking of the atomic structure of the varphi29 connector into the three-dimensional reconstruction of T7 connector reveals that the narrow, distal region of the two oligomers are almost identical. This region of the varphi29 connector has been suggested to be involved in DNA translocation, and is composed of an alpha-beta-alpha-beta-beta-alpha motif. A search for alpha-helices in the same region of the T7 three-dimensional map has located three alpha-helices in approximately the same position as those of the varphi29 connector. A comparison of the predicted secondary structure of several bacteriophage connectors, including among others T7, varphi29, P22 and SPP1, reveals that, despite the lack of sequence homology, they seem to contain the same alpha-beta-alpha-beta-beta-alpha motif as that present in the varphi29 connector. These results allow us to suggest a common architecture related to a basic component of the DNA translocating machinery for several viruses.