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Bacteriophages (viruses of bacteria) use a specialized organelle called a tail to deliver their genetic material and proteins across the cell envelope during infection (1-4). In phages with the most [...], Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post- host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

Author(s): Mikhail M. Shneider [sup.1] [sup.2] , Sergey A. Buth [sup.1] , Brian T. Ho [sup.3] , Marek Basler [sup.3] , John J. Mekalanos [sup.3] , Petr G. Leiman [sup.1] [...], An X-ray structure of bacterial type VI secretion system components reveals that PAAR family proteins bind at the tip of the VgrG spike, providing new insights into the mechanisms of type VI secretion; experiments using bacteria confirmed the importance of PAAR proteins. A mechanism for T6SS translocation The bacterial type VI secretion system (T6SS) is an organelle responsible for translocating a range of toxic effector molecules into both bacterial and eukaryotic prey cells. This manuscript describes how proteins from the PAAR-repeat superfamily form a sharp conical extension on the VgrG bacteriophage tail spike, a protein complex involved in penetrating the prey cell, and also have a role in recruiting effectors. These data support a new model for the T6SS in which a sharpened spike is decorated by multiple effectors that are delivered in concert into target cells by a single contraction-driven translocation event. The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells.sup.1,2,3,4,5. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike.sup.6,7. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.

Author(s): Shuji Kanamaru [1, 4]; Petr G. Leiman [1, 4]; Victor A. Kostyuchenko [1, 2]; Paul R. Chipman [1]; Vadim V. Mesyanzhinov [2]; Fumio Arisaka [3]; Michael G. Rossmann (corresponding [...]

Author(s): Alan A. Simpson [1, 2]; Yizhi Tao [1, 2, 3]; Petr G. Leiman [1]; Mohammed O. Badasso [4]; Yongning He [1]; Paul J. Jardine [5]; Norman H. Olson [1]; [...]

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. [ABSTRACT FROM AUTHOR]

Abstract Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.

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. [ABSTRACT FROM AUTHOR]

Summary: Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike‐shaped protein complex at its tip. The spike complex forms the centerpiece of a baseplate complex that terminates the sheath and the tube. The baseplate anchors the tail to the target cell membrane with the help of fibrous proteins emanating from it and triggers contraction of the sheath. The contracting sheath drives the tube with its spiky tip through the target cell membrane. Subsequently, the bacteriophage genome is injected through the tube. The structural transformation of the bacteriophage T4 baseplate upon binding to the host cell has been recently described in near‐atomic detail. In this review we discuss structural elements and features of this mechanism that are likely to be conserved in all contractile injection systems (systems evolutionary and structurally related to contractile bacteriophage tails). These include the type VI secretion system (T6SS), which is used by bacteria to transfer effectors into other bacteria and into eukaryotic cells, and tailocins, a large family of contractile bacteriophage tail‐like compounds that includes the P. aeruginosa R‐type pyocins. [ABSTRACT FROM AUTHOR]