Biology subjects

Teschke, C. M.

Publications and source records attributed to Teschke, C. M..

5 recordsLinked to original sources

bioRxiv2024

Structural Model of Bacteriophage P22 Scaffolding Protein in a Procapsid by Magic-Angle Spinning NMR

Icosahedral dsDNA viruses such as the tailed bacteriophages and herpesviruses have a conserved pathway to virion assembly that is initiated from a scaffolding protein driven procapsid formation. The dsDNA is actively packaged into procapsids, which undergo complex maturation reactions to form infectious virions. In bacteriophage P22, scaffolding protein (SP) directs the assembly of coat proteins into procapsids that have a T=7 icosahedral arrangement, en route to the formation of the mature P22 capsid. Other than the C-terminal helix-turn-helix involved in interaction with coat protein, the structure of the P22 303 amino acid scaffolding protein within the procapsid is not understood. Here, we present a structural model of P22 scaffolding protein encapsulated within the 23 MDa procapsid determined by magic angle spinning NMR spectroscopy. We took advantage of the 10-fold sensitivity gains afforded by the novel CPMAS CryoProbe to establish the secondary structure of P22 scaffolding protein and employed 19F MAS NMR experiments to probe its oligomeric state in the procapsid. Our results indicate that the scaffolding protein has both -helical and disordered segments and forms a trimer of dimers when bound to the procapsid lattice. This work provides the first structural information for P22 SP beyond the C-terminal helix-turn-helix and demonstrates the power of MAS NMR to understand higher-order viral protein assemblies involving structural components that are inaccessible to other structural biology techniques.

biochemistry↗

bioRxiv2024

Templated trimerization of the phage L decoration protein on capsids

The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a three-stranded intertwined spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec1-98) was used to demonstrate that the stable globular OB-fold domain folds independently of the C-terminal residues. However, Dec1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, however, implying Dec trimer assembly in vivo is templated by the phage capsid. The Thermodynamic Hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid.

biochemistry↗

bioRxiv2023

Bacteriophage P22 SieA mediated superinfection exclusion

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC, sieA, and sieB genes. Here we report that the P22-encoded SieA protein is the only phage protein required for exclusion by the SieA system, and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants were isolated that overcome the SieA block, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in target specificity. Our data are consistent with a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit. HIGHLIGHTSO_LIPhage P22 SieA protein blocks DNA injection by P22-like phages C_LIO_LISieA is an inner membrane protein C_LIO_LIHybrid P22 phages with phage HK620 ejection proteins partially escape SieA exclusion C_LIO_LISieA escape mutants of P22 alter the gp16 and 20 proteins that form the DNA ejection tube. C_LI

biochemistry↗

bioRxiv2022

Identification of a second site for coat protein binding in bacteriophage P22 scaffolding protein

Scaffolding proteins are essential for the assembly of most tailed, double-stranded DNA bacteriophages as well as herpesviruses. These proteins interact specifically with the coat proteins to efficiently assemble procapsids with the correct morphology. A helix-turn-helix (HTH) domain of bacteriophage P22 scaffolding protein is essential for coat binding, but the presence of additional coat protein binding sites has been predicted. An alanine substitution at scaffolding protein residue L245 causes a strong cold-sensitive phenotype. Both in vivo and in vitro assembly with L245A scaffolding protein yields aberrant and petite particles at non-permissive temperatures. The L245A scaffolding protein is destabilized as determined by thermal melts monitored by circular dichroism. Through crosslinking studies, residue L245 interacts with the coat protein A-domain residue D198, which has been predicted previously to contain a scaffolding protein binding site. L245 also binds R101 in the coat protein P-domain as well as E18 in the N-arm. These results demonstrate the presence of secondary coat binding sites that may function in conjunction with the HTH domain to promote the assembly of procapsids with the correct curvature. ImportanceMany dsDNA viruses, including tailed bacteriophages and Herpesviruses, assemble precursor capsids, or procapsids, using an essential catalytic scaffolding protein. How scaffolding proteins induce proper assembly of their major capsid proteins remains unclear. The scaffolding protein of bacteriophage P22 has a C-terminal helix-turn-helix domain that interacts with the N-arm of the coat protein to activate it for assembly. Here, a second potential coat protein interaction site is identified in scaffolding protein at residue L245. Residue L245 may be involved in stabilizing a small helical domain based the effect of the substitution scaffolding protein stability and a predicted model of the scaffolding protein fold, thereby indicating additional complexity in the interaction between coat and scaffolding proteins.

biochemistry↗

bioRxiv2018

How phage L capsid decoration protein distinguishes between nearly identical binding sites on an icosahedral virion

The major coat proteins of dsDNA tailed phages and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary "decoration" (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that precisely distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. Lastly, the NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

biophysics↗