Effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein

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, Histagged 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 ␣-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 metastable 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.

Section: Discussionmentioning

confidence: 99%

Structural Transformations Accompanying the Assembly of Bacteriophage P22 Portal Protein Rings in Vitro

Moore¹,

Prevelige

2001

“…SDS-PAGE, Western blot, and protein concentration determination were carried out as described previously (12). Analytical size exclusion chromatography was carried out in a prepacked Superose 6 column (Amersham Biosciences AB) calibrated and run as described previously (16). The Stokes radius derived from the K av value of YueB780 was determined by extrapolation from a plot of Stokes radii of standard proteins versus (Ϫlog…”

Section: Purification Of the His 6 -Tagged Receptor Version Yueb780 Amentioning

confidence: 99%

“…where V e is the elution volume of the protein, V 0 is the column void volume, and V t is the column total volume accessible to solvent (16). The standard proteins used were thyroglobulin (Stokes radius ϭ 8.5 nm), ovalbumin (3.05 nm), and myoglobin (2.07 nm).…”

Section: Purification Of the His 6 -Tagged Receptor Version Yueb780 Amentioning

confidence: 99%

The Ectodomain of the Viral Receptor YueB Forms a Fiber That Triggers Ejection of Bacteriophage SPP1 DNA

São-José

Lhuillier

Lurz

et al. 2006

Self Cite

128

The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37°C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15°C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.

“…Purified gp6 cross-linked with 25 mM glutaraldehyde was used as the control for electrophoretic mobility of covalently bound gp6 oligomers (27). Air oxidation was sufficient for the formation of disulfide bridges.…”

Section: Engineering Of Intersubunit Disulfide Bridges In Spp1 Portalmentioning

confidence: 99%

Structural Rearrangements between Portal Protein Subunits Are Essential for Viral DNA Translocation

Cuervo

Vaney

Antson

et al. 2007

Self Cite

Transport of DNA into preformed procapsids is a general strategy for genome packing inside virus particles. In most viruses, this task is accomplished by a complex of the viral packaging ATPase with the portal protein assembled at a specialized vertex of the procapsid. Such molecular motor translocates DNA through the central tunnel of the portal protein. A central question to understand this mechanism is whether the portal is a mere conduit for DNA or whether it participates actively on DNA translocation. The most constricted part of the bacteriophage SPP1 portal tunnel is formed by twelve loops, each contributed from one individual subunit. The position of each loop is stabilized by interactions with helix ␣-5, which extends into the portal putative ATPase docking interface. Here, we have engineered intersubunit disulfide bridges between ␣-5s of adjacent portal ring subunits. Such covalent constraint blocked DNA packaging, whereas reduction of the disulfide bridges restored normal packaging activity. DNA exit through the portal in SPP1 virions was unaffected. The data demonstrate that mobility between ␣-5 helices is essential for the mechanism of viral DNA translocation. We propose that the ␣-5 structural rearrangements serve to coordinate ATPase activity with the positions of portal tunnel loops relative to the DNA double helix.Portal proteins are hollow oligomers localized asymmetrically at a single vertex of icosahedral capsids of tailed bacteriophages and herpesviruses (1, 2) providing a tunnel for doublestranded DNA entry and exit (see Fig. 1, A-C) (5-7). The viral genome is packaged into procapsids by a DNA translocation motor that assembles at the portal vertex of the procapsid structure (step from states I to II of Fig. 1A). The motor is composed of the portal protein, an ATPase (large terminase subunit), and a third component (small terminase subunit or a pRNA) (8). How this large complex uses ATP hydrolysis (9 -13) to pump double-stranded DNA against a steep concentration gradient remains a mystery. Although it is well established that the ATPase energetically fuels DNA translocation, biochemical and genetic evidence shows also that the portal protein regulates ATPase activity (13) and that portal mutations impair DNA packaging (13-16). However, a mechanistic role of the portal protein and the function of its cross-talk with the terminase in the DNA packaging reaction remain to be demonstrated. X-ray structures of portal proteins from bacteriophages phi29 (p10) (5) and SPP1 (gp6) (17) show that they share a very similar structure. Their most unusual feature is the presence of a prominent distortion in helix ␣6. Helix ␣6 of the SPP1 portal presents a 136°kink stabilized by hydrogen bonding with crown residues and by van der Waals interactions with the carboxyl terminus of helix ␣5 (see Fig. 1C) (17). In isolated gp6, a 13-mer, helices ␣5 and ␣6 are connected by a loop that protrudes to the portal tunnel interior (17). This region is disorganized in the phi29 portal structure (5).The gp6 form found in ...