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.     

Solution structure and dynamics of a heat shock protein assembly probed by hydrogen exchange and mass spectrometry

The solution conformation and dynamics of the 16.9 kDa small heat shock protein from wheat have been studied using a combination of hydrogen/deuterium exchange, proteolytic digestion, and mass spectrometry. At room temperature, HSP16.9 exists as a dodecameric assembly. Regions of HSP16.9 that form extensive and essential intersubunit contacts in the assembly, including residues 1-40 and 131-151, show little or no protection against hydrogen/deuterium exchange after incubation in D(2)O for 5 s. The high levels of hydrogen/deuterium exchange indicate that these regions have experienced large conformational fluctuations in solution, breaking intersubunit contacts and exposing buried amide hydrogens to solvent. When HSP16.9 is pulse labeled for 10 ms, residues 1-40 and 131-151 are substantially more protected than they are after 5 s. Thus, the breaking of intersubunit contacts occurs on a time scale between 10 milliseconds and 5 s. At 42 degrees C, HSP16.9 exists in a suboligomeric form. When the intrinsic temperature dependence of hydrogen/deuterium exchange is taken into account, exchange patterns at 25 and 42 degrees C are identical within experimental error, suggesting that the conformation of individual HSP16.9 subunits is the same in both the dodecameric and subdodecameric forms. Significant protection is seen in regions that form the dimeric interface, suggesting that the stable suboligomeric form is a dimer. Taken together, these results suggest that heat activation of HSP16.9 occurs by shifting the dodecamer <--> dimer equilibrium in favor of free dimers. The conformation of the dimers themselves does not appear to be altered with an increase in temperature.     

Structure of bacteriophage P22 portal protein in relation to assembly: investigation by Raman spectroscopy.

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.     

Binding-induced stabilization and assembly of the phage P22 tail accessory factor gp4

To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.     

Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging

The gene 1 protein of Salmonella bacteriophage P22 is located at the DNA packaging vertex of the mature particle. The protein is incorporated into the procapsid shell during shell assembly and is required for DNA packaging. The unassembled precursor form of the gene 1 protein has been purified from cells infected with mutants blocked in procapsid assembly. The purified 90,000-dalton protein was dimeric or monomeric; upon storage in the cold it formed 20S cyclic dodecamers. Computer filtering of negatively stained electron micrographs revealed 12 arms and knobs projecting from a central ring, with a 30-A channel at the center. Similar dodecameric rings were released from disrupted procapsid shells. These results indicate that the gene 1 protein is organized as a cyclic dodecamer within the procapsid shell and serves as the portal through which P22 DNA is threaded during DNA packaging. The presence of a 12-fold ring located at a 5-fold portal vertex appears to be a conserved structural theme of the DNA packaging apparatus of double-stranded DNA phages.     

Structural framework for DNA translocation via the viral portal protein

Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal-DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a 'mexican wave' of positional and conformational changes propagating sequentially along this belt.     

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.     

High-sensitivity mass spectrometry for imaging subunit interactions: hydrogen/deuterium exchange

In recent years, advances in mass spectrometry have provided unprecedented knowledge of protein expression within cells. It has become apparent that many proteins function as macromolecular complexes. Structural genomics programs are determining the fold of these proteins at an increasing rate and electron microscopic tomography potentially provides a means to determine the location of these complexes within the cell. A complete understanding of the molecular mechanism of these proteins requires detailed information on the interactions and dynamics within the complex. Recent advances in mass spectrometry now make it possible to use hydrogen/deuterium exchange to detect intersubunit interfaces and dynamics within supramolecular complexes.     

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.     

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme

Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.     

Portal-large terminase interactions of the bacteriophage T4 DNA packaging machine implicate a molecular lever mechanism for coupling ATPase to DNA translocation

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.     

Molecular genetics of bacteriophage P22 scaffolding protein's functional domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.     

Novel second-site suppression of a cold-sensitive defect in phage P22 procapsid assembly

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene 1 protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein. The 1-csH137 mutation prevents the incorporation of the portal protein into procapsids. In a mixed infection with cs+ phage, the mutant subunits are able to form functional portals, suggesting that the cold-sensitivity does not affect portal-portal interactions, but affects the interaction of portal subunits with some other molecular species involved in the initiation of portal assembly. Interestingly, the cs defect is suppressed by temperature-sensitive folding mutations at four sites in the P22 tailspike gene 9. The suppression is allele-specific; other tailspike tsf mutations fail to suppress the cs defect. Translation through a suppressor site is required for suppression. This observation is unexpected, since analysis of nonsense mutations in this gene indicates that it is not required for procapsid assembly. Examination of the nucleic acid sequences in the neighborhood of each of the suppressor sites shows significant sequence similarity with the scaffolding gene translational initiation region on the late message. This supports a previously proposed model, in which procapsid assembly is normally initiated in a region on the late messenger RNA that includes the gene 8 start site. By this model, the suppressor mutations may be acting through protein-RNA interactions, changing sequences that identify alternative or competing sites at which the mutant portal subunits may be organized for assembly into the differentiated vertex of the phage capsid.     

Hexuronates influence the oligomeric form of the Dps structural protein of bacterial nucleoid and its ability to bind to linear DNA fragments

Proteins of the Dps family perform a dual function in bacterial cells. As ferritins, they protect cells from destructive effects of Fe²⁺ ions, while interacting with DNA they condense the genome in the absence of nutrients. The ability of Dps to self-aggregate is of a great importance. The way of genome remodelling from the condensed state to the active one is not yet known. Here, the effects of two sugar ligands on Dps interaction with DNA have been studied in vitro. For the first time it was demonstrated that D-glucuronate and D-galacturonate, but not D-glucose, can decompose the dodecameric structure of the protein and D-glucuronate stimulated the formation of binary complexes with the linear DNA fragments. As a result of flexible molecular docking, it was found that the molecules of all three sugars potentially can form clusters inside the protein cavity of Dps, but D-glucuronate and D-galacturonate were also bound in the region of intersubunit contacts of oligomer. The consequent destabilization of the intersubunit bonding network can, thus, be the main factor provoking the protein decay to the smaller oligomeric forms. Such a structural rearrangement, leading to a reduction in aggregation, may play a key role in genome decondensation during cell transition to the phase of rapid growth.     

An ATP hydrolysis sensor in the DNA packaging motor from bacteriophage T4 suggests an inchworm-type translocation mechanism

Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.     

Allosteric signaling in the biotin repressor occurs via local folding coupled to global dampening of protein dynamics

The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5′-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding.     

Raman spectroscopy and deuterium exchange of the filamentous phage fd

The filamentous phage fd has been investigated using the techniques of Raman spectroscopy and deuterium exchange. Despite the rather uniform secondary structure of the fd phage coat protein, which is predominantly alpha-helix, the deuterium exchange is complex. A substantial fraction of the helical peptides exchange deuterium by 8 h at room temperature, yet another substantial fraction does not exchange following an additional 5 months at 4 degrees C. Heating the phage to 70 degrees C for several hours leads to additional deuterium exchange compared to samples soaked for 5 months in heavy water. We suggest that the wide variation in peptide exchange rates may be related to the phage protein quaternary structure, which has been shown to be a double layer of tightly packed helices. The accomplishment of enhanced exchange by reaction at high temperature combined with digital difference spectroscopic methods has enabled us to define the structure of the amide III and III' bands. The complexity of these bands is unexpected for a simple helical protein, but we suggest that the complexity arises at least in part from end-effects that become important in short alpha-helices.     

Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.