Preliminary crystallographic analysis of the bacteriophage P22 portal protein

Portal proteins are components of large oligomeric dsDNA pumps connecting the icosahedral capsid of tailed bacteriophages to the tail. Prior to the tail attachment, dsDNA is actively pumped through a central cavity formed by the subunits. We have studied the portal protein of bacteriophage P22, which is the largest connector characterized among the tailed bacteriophages. The molecular weight of the monomer is 82.7 kDa, and it spontaneously assembles into an oligomeric structure of approximately 1.0 MDa. Here we present a preliminary biochemical and crystallographic characterization of this large macromolecular complex. The main difficulties related to the crystallization of P22 portal protein lay in the intrinsic dynamic nature of the portal oligomer. Recombinant connectors assembled from portal monomers expressed in Escherichia coli form rings of different stoichiometry in solution, which cannot be separated on the basis of their size. To overcome this intrinsic heterogeneity we devised a biochemical purification that separates different ring populations on the basis of their charge. Small ordered crystals were grown from drops containing a high concentration of the kosmotropic agent tert-butanol and used for data collection. A preliminary crystallographic analysis to 7.0-A resolution revealed that the P22 portal protein crystallized in space group I4 with unit cell dimensions a=b=409.4A, c=260.4A. This unit cell contains a total of eight connectors. Analysis of the noncrystallographic symmetry by the self-rotation function unambiguously confirmed that bacteriophage P22 portal protein is a dodecamer with a periodicity of 30 degrees. The cryo-EM reconstruction of the dodecahedral bacteriophage T3 portal protein will be used as a model to initiate phase extension and structure determination.     

Solution x-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.     

Cloning, purification, and preliminary characterization by circular dichroism and NMR of a carboxyl-terminal domain of the bacteriophage P22 scaffolding protein.

Assembly of double-stranded DNA viruses and bacteriophages involves the polymerization of several hundred molecules of coat protein, directed by an internal scaffolding protein. A 163-amino acid carboxyl-terminal fragment of the 303-amino acid bacteriophage P22 scaffolding protein was cloned, overexpressed, and purified. This fragment is active in procapsid assembly reactions in vitro. The circular dichroism spectrum of the fragment, as well as the 1D-NMR and 15N-1H HSQC spectra of the uniformly-labeled protein, indicate that stable secondary structure elements are present. Determination of the three dimensional packing of these elements into the folded scaffolding protein fragment is underway. Structure-based drug design targeted at structural proteins required for viral assembly may have potential as a therapeutic strategy.     

An aggregation-prone intermediate species is present in the unfolding pathway of the monomeric portal protein of bacteriophage P22: implications for portal assembly

The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.     

New Technology for Estimating Seed Production of Moist-Soil Plants

ABSTRACT Waterfowl biologists estimate seed production in moist-soil wetlands to calculate duck-energy days (DEDs) and evaluate management techniques. Previously developed models that predict plant seed yield using morphological measurements are tedious and time consuming. We developed simple linear regression models that indirectly and directly related seed-head area to seed production for 7 common moist-soil plants using portable and desktop scanners and a dot grid, and compared time spent processing samples and predictive ability among models. To construct models, we randomly collected approximately 60 plants/species at the Tennessee National Wildlife Refuge, USA, during September 2005 and 2006, threshed and dried seed from seed heads, and related dry mass to seed-head area. All models explained substantial variation in seed mass (R²< 0.87) and had high predictive ability (R²_predicted < 0.84). Processing time of seed heads averaged 22 and 3 times longer for the dot grid and portable scanner, respectively, than for the desktop scanner. We recommend use of desktop scanners for accurate and rapid estimation of moist-soil plant seed production. Seed predictions per plant from our models can be used to estimate total seed production and DEDs in moist-soil wetlands.     

A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation

The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a φ29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.The DNA packaging motor of double-stranded DNA bacteriophages translocates genomic DNA into a preformed procapsid to near crystalline density and is the strongest motor characterized to date. The packaging motor of the Bacillus subtilis phage φ29 can work against 57 piconewtons of internal force and translocate 2 bp of DNA per ATP hydrolyzed at a maximum velocity of 103 bp/s (1, 2). The motor complex is assembled on a dodecamer of the connector protein, which replaces a pentameric vertex in the procapsid and serves both as a portal for DNA passage and the docking site for the other packaging components (3).To successfully package a full-length genome, incorporation of one and only one connector vertex is essential (4). In vivo, nearly every assembled procapsid has one and only one connector vertex and is able to package DNA and mature into an infectious phage (5). This narrow distribution in which 95% of particles have a single connector vertex cannot be explained by random statistical incorporation. The control mechanism is coupled to the procapsid assembly process. Procapsid assembly requires the copolymerization of hundreds of copies each of the capsid and scaffolding proteins as well as a dodecamer of the portal or connector protein. The scaffolding protein acts to both activate the coat protein for assembly and ensure proper form determination. In the absence of scaffolding protein, uncontrolled polymerization results in the assembly of aberrant structures. In a properly assembled procapsid, the portal protein is located at one vertex, whereas scaffolding protein occupies the bulk of the interior space and is subsequently removed during DNA packaging by either proteolysis or simple release. Mutational studies have indicated that scaffolding protein is involved either directly or indirectly in the incorporation of the connector vertex during procapsid assembly in a variety of phages (6–8).In φ29, the connector vertex is specifically incorporated at one of the two 5-fold vertices lying on the long axis of a prolate procapsid composed of 235 copies of capsid protein and containing ∼180 copies of scaffolding protein (9, 10). The structure of the 33-kDa connector protein subunit consists of three long central α-helices bridging wide and narrow domains that are rich in β-sheets and extended polypeptides (Fig. 1A) (10–12). The 12 subunits are arranged to form a 75-Å-long tapered grommet-shaped structure with an external diameter of 69 Å at the wide end and 33 Å at the narrow end. By fitting the crystal structure of the connector dodecamer into the cryo-EM¹ density of the procapsid, the orientation of connector at the unique vertex of the procapsid was revealed. The wide domain of connector protein lies inside the procapsid, and the narrow domain is exposed to the exterior and makes contacts with the other parts of the motor complex (11). The 11-kDa scaffolding protein subunits form nanomolar affinity homodimers resembling arrows in solution. Each subunit contributes one side of the arrowhead and one-half of the long coiled coil shaft (Fig. 1B) (13). The subunit structure consists of three helical segments. A three-turn N-terminal helix (α1) followed by a five-residue loop, and an antiparallel five-turn helix (α2) makes up the arrowhead and part of the proximal part of the shaft. A three-residue loop and a seven-turn helix (α3) complete the shaft. The C-terminal 15 residues, which interact with capsid protein as determined in the in vitro assembly assay, are disordered in the crystal structure (14).Open in a separate windowFig. 1.The x-ray crystal structures of connector protein (Protein Data Bank code 1FOU, chains A and B) (A) and scaffolding protein (Protein Data Bank code 1NO4, chains A and B) (B).We have recently reported the development of an in vitro assembly system for phage φ29 in which purified connector protein complex can be successfully incorporated (15). The addition of connector protein dodecamers to coat and scaffolding subunits accelerated the rate of assembly and lowered the critical concentration, suggesting involvement in nucleation of assembly (15). Here we used native mass spectrometry, chemical cross-linking, and mutational analysis to characterize the interactions between the connector and the scaffolding proteins and develop a model of the scaffolding-connector complex, which provides a molecular model of how scaffolding protein might mediate stringent incorporation of one and only one connector dodecamer.     

Identification and characterization of the domain structure of bacteriophage P22 coat protein.

The bacteriophage P22 serves as a model for assembly of icosahedral dsDNA viruses. The P22 procapsid, which constitutes the precursor for DNA packaging, is built from 420 copies of a single coat protein with the aid of stoichiometric amounts of scaffolding protein. Upon DNA entry, the procapsid shell expands and matures into a stable virion. It was proposed that expansion is mediated by hinge bending and domain movement. We have used limited proteolysis to map the dynamic stability of the coat protein domain structures. The coat protein monomer is susceptible to proteolytic digestion, but limited proteolysis by small quantities of elastase or chymotrypsin yielded two metastable fragments (domains). The N-terminal domain (residues 1-180) is linked to the C-terminal domain (residues 205-429) by a protease-susceptible loop (residues 180-205). The two domains remain associated after the loop cleavage. Although only a small change of secondary structure results from the loop cleavage, both tertiary interdomain contacts and subunit thermostability are diminished. The intact loop is also required for assembly of the monomeric coat protein into procapsids. Upon assembly, coat protein becomes largely protease-resistant, baring cleavage within the loop region of about half of the subunits. Loop cleavage decreases the stability of the procapsids and facilitates heat-induced shell expansion. Upon expansion, the loop becomes protease-resistant. Our data suggest the loop region becomes more ordered during assembly and maturation and thereby plays an important role in both of these stages.     

Niche modelling of the Chilean recluse spider Loxosceles laeta and araneophagic spitting spider Scytodes globula and risk for loxoscelism in Chile

In Chile, all necrotic arachnidism is attributed to the Chilean recluse spider Loxosceles laeta (Nicolet) (Araneae: Sicariidae). It is predated by the spitting spider Scytodes globula (Nicolet) (Araneae: Scytodidae). The biology of each of these species is not well known and it is important to clarify their distributions. The aims of this study are to elucidate the variables involved in the niches of both species based on environmental and human footprint variables, and to construct geographic maps that will be useful in estimating potential distributions and in defining a map of estimated risk for loxoscelism in Chile. Loxosceles laeta was found to be associated with high temperatures and low rates of precipitation, whereas although S. globula was also associated with high temperatures, its distribution was associated with a higher level of precipitation. The main variable associated with the distribution of L. laeta was the human footprint (48.6%), which suggests that this is a highly invasive species. Similarly to other species, the distribution of L. laeta reaches its southern limit at the Los Lagos region in Chile, which coincides with high levels of precipitation and low temperatures. The potential distribution of L. laeta in Chile corresponds to the distribution of cases of loxoscelism.     

A Retroviral Chimeric Capsid Protein Reveals the Role of the N-Terminal β-Hairpin in Mature Core Assembly

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.