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The stable ribonucleoprotein (RNP) complex formed between the Lactococcus lactis group II intron and its self-encoded LtrA protein is essential for the intron's genetic mobility. In this study, we report the biochemical, compositional, hydrodynamic and structural properties of active group II intron RNP particles (+A) isolated from its native host using a novel purification scheme. We employed small-angle X-ray scattering to determine the structural properties of these particles as they exist in solution. Using sucrose as a contrasting agent, we derived a two-phase quaternary model of the protein-RNA complex. This approach revealed that the spatial properties of the complex are largely defined by the RNA component, with the protein dimer located near the center of mass. A transfer RNA fusion engineered into domain II of the intron provided a distinct landmark consistent with this interpretation. Comparison of the derived +A RNP shape with that of the previously reported precursor intron (ΔA) particle extends previous findings that the loosely packed precursor RNP undergoes a dramatic conformational change as it compacts into its active form. Our results provide insights into the quaternary arrangement of these RNP complexes in solution, an important step to understanding the transition of the group II intron from the precursor to a species fully active for DNA invasion.

Glycobiology, 27 (5), ISSN:0959-6658

Bordetella Bacteriophage (BPP-1) infects both Bordetella bronchiseptica, and B. pertussis, the bacterium that causes whooping cough. BPP-1 is a short-tailed dsDNA virus and a member in the Podoviridae family. It has a T=7 icosahedral head with a diameter of ~700 A and a tail on one of the twelve icosahedral vertices [1]. The capsid head is composed of two proteins: a major capsid protein (MCP) and a cement protein, and neither has recognizable sequence similarity with proteins in other well studied dsDNA bacteriophages like HK97 and PRD1 [2, 3]. We have determined the structure of the icosahedral head of BPP-1 to 3.4A resolution using single particle cryo-electron microscopy (cryoEM) (Fig. 1a) and derived an atomic model of BPP-1. Briefly, cryoEM images were recorded on Kodak SO-163 micrographs at 59K magnification in an FEI Titan Krios microscope at 300kV with a dosage of ~25 e−2/A on the specimen. ~43,000 particles from 340 micrographs were selected for image processing and three dimensional (3D) reconstruction (Fig. 1b, c). Local averaging of the 7 copies of the cement or MCP protein in the asymmetric unit was performed to further improve resolution, model building was performed using Coot, and atomic models were refined using CCP4 (Fig. 1c–e) [4]. Fig. 1 Structure of BPP-1 at 3.4A resolution determined by cryoEM The BPP-1 head reconstruction has an angular shape with 420 MCP subunits making up the capsid shell and 210 dimers of the cement protein joining neighboring MCPs, together forming chainmail (Fig. 1c). Side chains of amino acids are clearly resolved in both proteins and were used for atomic model building. All but the last amino acid of the 140 amino acid residues of the cement protein were visible in the cryoEM density. The atomic model of the cement protein shows a jellyroll fold and a ~40A C-terminal arm (Fig. 1d). The interactions between the two monomers in the cement protein dimer are limited to the F-strand of the jellyroll (Fig. 1d), primarily through a pair of Pro residues, one on each monomer. In contrast, each cement protein monomer interacts with five underlying MCP molecules, mediated through loops of the jellyroll and through a β augmentation of the C-terminal arm (Fig. 1c). This use of the jellyroll fold for an auxiliary stabilizing role is quite remarkable because such fold has only been observed as a building block for viral capsids. Of the 331 residues of the BPP-1 MCP, we traced Thr6 to Val330 in our density map. The N and C termini are both located on the surface, but the first 5 and the last one amino acids are not visible, suggesting their flexibility (Fig. 1e). The BPP-1 MCP has the fold/architecture of the capsid protein of HK97 bacteriophage. We hereby divide it into three building blocks from N to C termini: chainmail-forming (cyan), β sheet-forming (purple) and capsomer-forming (green) (Fig. 1e) [2]. Surprisingly, the topologies of folding these three building blocks into this architecture are different between BPP-1 and HK97 (Fig. 1e, f): The last two of the three building blocks in gp5 of HK97 are swapped as compared to the sequence in BPP-1, in a rare, non-circular permutation, as found in DNA methyltransferases [5]. The difference in the topologies in folding suggests convergent evolution in selecting the characteristic HK97-like fold to build chainmail in the two viruses. It has been found that the internal pressure inside bacteriophages is very high due to the high packing density of the viral genome inside the viral capsid [6], and that different bacteriophages adopt distinctive strategies to stabilize their capsids to withstand the high pressure. HK97 uses covalent isopeptide bonds to link adjacent gp5 proteins to stabilize its capsid [2]. Having the same building blocks but lacking isopeptide bonds to join the chainmail-forming building blocks, BPP-1 uses an extra cement protein with the jellyroll fold to complete the chainmail (Fig. 1c). Our structure highlights the diverse strategies to build a highly stable dsDNA virus and suggests different maturation processes between the two bacteriophages.

The specific complex between the lambda phage OR3 operator and the Cro protein has been studied by proton NMR spectroscopy at 500 MHz. The DNA imino proton resonances of this complex have been assigned to specific base pairs using the known assignments of these resonances for the free operator. Increase of the protein/DNA ratio to complete saturation of the OR3 operator with the Cro protein made it possible to follow the shift changes of the resonances. Ambiguities were resolved by nuclear Overhauser effect measurements on the complex. The shifts of the imino proton resonance positions provide information on the changes induced in the conformation of the operator upon complex formation with a dimer of the Cro protein. The most striking shift occurs for the central (GC 9) base pair, which is known to have no direct contacts with the Cro protein. This shift may be induced by a bend in the OR3 operator DNA at the GC 9 base pair to accommodate the operator for the binding of the Cro protein dimer. The imino proton resonances of two additional base pairs can be observed in the complex, demonstrating an overall stabilization of the DNA structure by the binding of the Cro protein.

The formation of the interchain disulfide bonds in partially reduced Bence Jones proteins and immunoglobulins was studied in the presence of glutathione. It was found that only oxidized glutathione (GSSG) was effective for the formation of the interchain disulfide bonds in type gamma Bence Jones proteins and IgG. In type kappa Bence Jones proteins, on the other hand, no formation of the inter L-L disulfide bond was observed in the presence of GSSG at above pH 6. The kinetic pattern of disulfide bond formation of Bence Jones proteins was well interpreted by assuming that two monomers of a type gamma protein dimer are discriminated (monomers 1 and 2) and that only an intermediate in which the SH group on monomer 1 is blocked with GSSG can form a disulfide-bonded dimer and the intermediate in which the SH group on monomer 2 is blocked with GSSG can not. Comparison of the kinetic data for the formation of the interchain disulfide bonds of IgG with those for Bence Jones proteins suggested that H chain-GSSG mixed disulfide is a principal intermediate for the formation of the inter H-L disulfide bond.