An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano.

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.

Neck and capsid architecture of the robust Agrobacterium phage Milano.

Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of the Agrobacterium phage Milano. The Milano neck 1 protein connects the 12-fold symmetrical neck to a 5-fold vertex of the icosahedral capsid. Comparison of Milano neck 1 homologs leads to four proposed classes, likely evolved from the simplest one in siphophages to more complex ones in myo- and podophages. Milano neck is surrounded by the atypical collar, which covalently crosslinks the tail sheath to neck 1. The Milano capsid is decorated with three types of proteins, a minor capsid protein (mCP) and two linking proteins crosslinking the mCP to the major capsid protein. The extensive network of disulfide bonds within and between neck, collar, capsid and tail provides an exceptional structural stability to Milano.

Function of the bacteriophage P2 baseplate central spike Apex domain in the infection process.

The contractile tail of bacteriophage P2 functions to drive the tail tube across the outer membrane of its host bacterium, a prerequisite event for subsequent translocation of phage genomic DNA into the host cell. The tube is equipped with a spike-shaped protein (product of P2 gene V , gpV or Spike) that contains a membrane-attacking Apex domain carrying a centrally positioned Fe ion. The ion is enclosed in a histidine cage that is formed by three symmetry-related copies of a conserved HxH (histidine, any residue, histidine) sequence motif. Here, we used solution biophysics and X-ray crystallography to characterize the structure and properties of Spike mutants in which the Apex domain was either deleted or its histidine cage was either destroyed or replaced with a hydrophobic core. We found that the Apex domain is not required for the folding of full-length gpV or its middle intertwined ß-helical domain. Furthermore, despite its high conservation, the Apex domain is dispensable for infection in laboratory conditions. Collectively, our results show that the diameter of the Spike but not the nature of its Apex domain determines the efficiency of infection, which further strengthens the earlier hypothesis of a drill bit-like function of the Spike in host envelope disruption.

Structural basis of template strand deoxyuridine promoter recognition by a viral RNA polymerase.

Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the -10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the -10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host.

Computational models in the service of X-ray and cryo-electron microscopy structure determination.

Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a cryo-electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination.

Quantitative description of a contractile macromolecular machine.

Contractile injection systems (CISs) [type VI secretion system (T6SS), phage tails, and tailocins] use a contractile sheath-rigid tube machinery to breach cell walls and lipid membranes. The structures of the pre- and postcontraction states of several CISs are known, but the mechanism of contraction remains poorly understood. Combining structural information of the end states of the 12-megadalton R-type pyocin sheath-tube complex with thermodynamic and force spectroscopy analyses and an original modeling procedure, we describe the mechanism of pyocin contraction. We show that this nanomachine has an activation energy of 160 kilocalories/mole (kcal/mol), and it releases 2160 kcal/mol of heat and develops a force greater than 500 piconewtons. Our combined approach provides a quantitative and experimental description of the membrane penetration process by a CIS.

Reprogramming bacteriophage host range: design principles and strategies for engineering receptor binding proteins.

Bacteriophages (phages) use specialized tail machinery to deliver proteins and genetic material into a bacterial cell during infection. Attached at the distal ends of their tails are receptor binding proteins (RBPs) that recognize specific molecules exposed on host bacteria surfaces. Since the therapeutic capacity of naturally occurring phages is often limited by narrow host ranges, there is significant interest in expanding their host range via directed evolution or structure-guided engineering of their RBPs. Here, we describe the design principles of different RBP engineering platforms and draw attention to the mechanisms linking RBP binding and the correct spatial and temporal attachment of the phage to the bacterial surface. A deeper understanding of these mechanisms will directly benefit future engineering of more effective phage-based therapeutics.

Structure and function of virion RNA polymerase of a crAss-like phage.

CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.

The Central Spike Complex of Bacteriophage T4 Contacts PpiD in the Periplasm of Escherichia coli.

Infecting bacteriophage T4 uses a contractile tail structure to breach the envelope of the Escherichia coli host cell. During contraction, the tail tube headed with the "central spike complex" is thought to mechanically puncture the outer membrane. We show here that a purified tip fragment of the central spike complex interacts with periplasmic chaperone PpiD, which is anchored to the cytoplasmic membrane. PpiD may be involved in the penetration of the inner membrane by the T4 injection machinery, resulting in a DNA-conducting channel to translocate the phage DNA into the interior of the cell. Host cells with the ppiD gene deleted showed partial reduction in the plating efficiency of T4, suggesting a supporting role of PpiD to improve the efficiency of the infection process.

Structure and Function of the T4 Spackle Protein Gp61.3.

The bacteriophage T4 genome contains two genes that code for proteins with lysozyme activity-e and 5. Gene e encodes the well-known T4 lysozyme (commonly called T4L) that functions to break the peptidoglycan layer late in the infection cycle, which is required for liberating newly assembled phage progeny. Gene product 5 (gp5) is the tail-associated lysozyme, a component of the phage particle. It forms a spike at the tip of the tail tube and functions to pierce the outer membrane of the Escherichia coli host cell after the phage has attached to the cell surface. Gp5 contains a T4L-like lysozyme domain that locally digests the peptidoglycan layer upon infection. The T4 Spackle protein (encoded by gene 61.3) has been thought to play a role in the inhibition of gp5 lysozyme activity and, as a consequence, in making cells infected by bacteriophage T4 resistant to later infection by T4 and closely related phages. Here we show that (1) gp61.3 is secreted into the periplasm where its N-terminal periplasm-targeting peptide is cleaved off; (2) gp61.3 forms a 1:1 complex with the lysozyme domain of gp5 (gp5Lys); (3) gp61.3 selectively inhibits the activity of gp5, but not that of T4L; (4) overexpression of gp5 causes cell lysis. We also report a crystal structure of the gp61.3-gp5Lys complex that demonstrates that unlike other known lysozyme inhibitors, gp61.3 does not interact with the active site cleft. Instead, it forms a "wall" that blocks access of an extended polysaccharide substrate to the cleft and, possibly, locks the enzyme in an "open-jaw"-like conformation making catalysis impossible.

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7.

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

Action of a minimal contractile bactericidal nanomachine.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics1-4. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold1,2. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage5. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.

Structural basis for recognition of bacterial cell wall teichoic acid by pseudo-symmetric SH3b-like repeats of a viral peptidoglycan hydrolase.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 3'O-acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudo-symmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. Our multidisciplinary approach tackled an extremely challenging protein-glycopolymer complex and delineated a previously unknown recognition mechanism by which a phage endolysin specifically recognizes and targets WTA, suggesting an adaptable model for regulation of endolysin specificity.

Modeling the Architecture of Depolymerase-Containing Receptor Binding Proteins in Klebsiella Phages.

Klebsiella pneumoniae carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many Klebsiella bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two RBPs, but phages with up to 11 RBPs with depolymerase activity and a broad host spectrum have been identified. A detailed bioinformatic analysis shows that similar RBP domains repeatedly occur in K. pneumoniae phages with structural RBP domains for attachment of an RBP to the phage tail (anchor domain) or for branching of RBPs (T4gp10-like domain). Structural domains determining the RBP architecture are located at the N-terminus, while the depolymerase is located in the center of protein. Occasionally, the RBP is complemented with an autocleavable chaperone domain at the distal end serving for folding and multimerization. The enzymatic domain is subjected to an intense horizontal transfer to rapidly shift the phage host spectrum without affecting the RBP architecture. These analyses allowed to model a set of conserved RBP architectures, indicating evolutionary linkages.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.

Structure and transformation of bacteriophage A511 baseplate and tail upon infection of Listeria cells.

Contractile injection systems (bacteriophage tails, type VI secretions system, R-type pyocins, etc.) utilize a rigid tube/contractile sheath assembly for breaching the envelope of bacterial and eukaryotic cells. Among contractile injection systems, bacteriophages that infect Gram-positive bacteria represent the least understood members. Here, we describe the structure of Listeria bacteriophage A511 tail in its pre- and post-host attachment states (extended and contracted, respectively) using cryo-electron microscopy, cryo-electron tomography, and X-ray crystallography. We show that the structure of the tube-baseplate complex of A511 is similar to that of phage T4, but the A511 baseplate is decorated with different receptor-binding proteins, which undergo a large structural transformation upon host attachment and switch the symmetry of the baseplate-tail fiber assembly from threefold to sixfold. For the first time under native conditions, we show that contraction of the phage tail sheath assembly starts at the baseplate and propagates through the sheath in a domino-like motion.

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition.

The ability of phages to infect specific bacteria has led to their exploitation as bio-tools for bacterial remediation and detection. Many phages recognize bacterial hosts via adhesin tips of their long tail fibers (LTFs). Adhesin sequence plasticity modulates receptor specificity, and thus primarily defines a phage's host range. Here we present the crystal structure of an adhesin (gp38) attached to a trimeric ß-helical tip (gp37) from the Salmonella phage S16 LTF. Gp38 contains rare polyglycine type II helices folded into a packed lattice, herein designated "PGII sandwich." Sequence variability within the domain is limited to surface-exposed helices and distal loops that form putative receptor-binding sites. In silico analyses revealed a prevalence of the adhesin architecture among T-even phages, excluding the archetypal T4 phage. Overall, S16 LTF provides a valuable model for understanding binding mechanisms of phage adhesins, and for engineering of phage adhesins with expandable or modulated host ranges.

Structure and Analysis of R1 and R2 Pyocin Receptor-Binding Fibers.

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to "simple" contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains-two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction.