A dimer as a building block in assembling RNA. A hexamer that gears bacterial virus phi29 DNA-translocating machinery

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a check mark outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg(2+), Ca(2+), and Mn(2+). Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.     

Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.     

Sequence requirement for hand-in-hand interaction in formation of RNA dimers and hexamers to gear phi29 DNA translocation motor.

Translocation of DNA or RNA is a ubiquitous phenomenon. One intricate translocation process is viral DNA packaging. During maturation, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into a limited space within the procapsid. We have revealed that phi29 DNA packaging is accomplished by a mechanism similar to driving a bolt with a hex nut, which consists of six DNA-packaging pRNAs. Four bases in each of the two pRNA loops are involved in RNA/RNA interactions to form a hexagonal complex that gears the DNA translocating machine. Without considering the tertiary interaction, in some cases only two G/C pairs between the interacting loops could provide certain pRNAs with activity. When all four bases were paired, at least one G/C pair was required for DNA packaging. The maximum number of base pairings between the two loops to allow pRNA to retain wild-type activity was five, whereas the minimum number was five for one loop and three for the other. The findings were supported by phylogenetic analysis of seven pRNAs from different phages. A 75-base RNA segment, bases 23-97, was able to form dimer, to interlock into the hexamer, to compete with full-length pRNA for procapsid binding, and therefore to inhibit phi29 assembly in vitro. Our result suggests that segment 23-97 is a self-folded, independent domain involved in procapsid binding and RNA/RNA interaction in dimer and hexamer formation, whereas bases 1-22 and 98-120 are involved in DNA translocation but dispensable for RNA/RNA interaction. Therefore, this 75-base RNA could be a model for structural studies in RNA dimerization.     

Mapping the inter-RNA interaction of bacterial virus phi29 packaging RNA by site-specific photoaffinity cross-linking

During replication, the lengthy genome of double-stranded DNA viruses is translocated with remarkable velocity into a limited space within the procapsid. The question of how this fascinating task is accomplished has long been a puzzle. Our recent investigation suggests that phi29 DNA packaging is accomplished by a mechanism similar to the driving of a bolt with a hex nut and that six packaging RNAs (pRNAs) form a hexagonal complex to gear the DNA-translocating machine (Chen, C., and Guo, P. (1997) J. Virol. 71, 3864-3871; Zhang, F., Lemieux, S., Wu, X., St.-Arnaud, S., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell 2, 141-147; Guo, P., Zhang, C., Chen, C., Garver, K., and Trottier, M., (1998) Mol. Cell 2, 149-155). In the current study, circularly permuted pRNAs were used to position an azidophenacyl photoreactive cross-linking agent specifically at a strategic site that was predicted to be involved in pRNA-pRNA interaction. Cross-linked pRNA dimers were isolated, and the sites of cross-link were mapped by primer extension. The cross-linked pRNA dimer retained full activity in phi29 procapsid binding and genomic DNA translocation, indicating that the cross-link distance constraints identified in dimer formation reflect the native pRNA complex. Both cross-linked dimers either containing or not containing the interlocking loops for programmed hexamer formation bound procapsid equally well; however, only the one containing the interlocking loops programmed for hexamer formation was active in phi29 DNA packaging. The cross-linked pRNA dimers were also identified as the minimum binding unit necessary for procapsid binding. Primer extension of the purified cross-linked pRNA dimers revealed that base G(82) was cross-linked to bases G(39), G(40), A(41), C(49), G(62), C(63), and C(64), which contribute to the formation of the three-way junction, suggesting that these bases are proximate in the formation of pRNA tertiary structure. Interestingly, the photoaffinity agent in the left interacting loop did not cross-link directly to the right loop as expected but cross-linked to bases adjacent to the right loop. These data provide a background for future modeling of pRNA tertiary structure.     

The procapsid binding domain of phi29 packaging RNA has a modular architecture and requires 2'-hydroxyl groups in packaging RNA interaction

The phi29 packaging RNA (pRNA) is an essential component in the phi29 bacteriophage DNA packaging motor, the strongest biomolecular motor known today. Utilizing Mg2+-dependent intermolecular base pairing interactions between two 4-nucleotide loops within the pRNA procapsid binding domain, multiple copies of pRNA form a ring-shaped complex that is indispensable for packaging motor function. To understand pRNA structural organization and pRNA/pRNA interaction, studies were carried out on pRNA closed dimers, the simplest functional pRNA complex believed to be the building blocks for assembling the oligomeric ring. Tertiary folding and interactions in various pRNA mutants were evaluated based on measured closed dimer affinity that is directly linked to the proper positioning of the interacting loops. The data revealed that the procapsid binding domain contains two autonomous modules that are capable of interacting noncovalently to form a fully active species in pRNA/pRNA interaction. Deleting the 2'-hydroxyl groups in one of the interacting loops weakens the dimer affinity by 125-fold, suggesting potential tertiary interactions involving these 2'-hydroxyl groups. The results provide evidence that nonbase functional groups are involved in pRNA folding and interaction and lead to a simple model that describes the pRNA monomer configuration in terms of three arms spanning a hinge. The functional constructs developed here will aid biophysical and biochemical investigations of pRNA structure and function, as well as developments of pRNA-based technology for nanoscience and gene therapy.     

Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization

An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.     

RD-114, baboon, and woolly monkey viral RNA's compared in size and structure.

The molecular weights, subunit compositions, and secondary structure patterns of the RNAs from an endogenous baboon virus and from a woolly monkey sarcoma virus were examined and compared to the properties of the RNA of RD-114, an endogenous feline virus. The high molecular weight RNA extracted from each of these three viruses has a sedimentation coefficient of 52S, and a molecular length, measured by electron microscopy, of 16-20 kb (kb=kilobase, 1000 nucleotides). Each such RNA is a dimer, containing two monomer subunits of 8-10 kb in length (molecular weight 3 X 10(6) daltons). The two monomer subunits are joined at their non-poly(A) ends in a structure called the dimer linkage structure. The appearance of this structure is somewhat different for the different viruses. The dimer linkage dissociates at temperature estimated to be 87 degrees C in aqueous 0.1M Na+ for RD-114 and baboon viral RNAs, but at the lower temperature of 66 degrees C for woolly monkey RNA. All three viral RNAs have two large loops of similar size and position symmetrically placed on either side of the dimer linkage structure. Since the baboon virus is partially related to RD-114, and the woolly monkey virus is unrelated to either of the other two, the dimer linkage and symmetrical loops are surprisingly similar and may well be common features of type C virus RNAs.     

Computer modeling of three-dimensional structure of DNA-packaging RNA (pRNA) monomer, dimer, and hexamer of Phi29 DNA packaging motor

A striking common feature in the maturation of all linear double-stranded DNA viruses is that their lengthy genome is translocated with remarkable velocity into the limited space within a preformed protein shell and packaged into near crystalline density. A DNA-translocating motor, powered by ATP hydrolysis, accomplishes this task, which would otherwise be energetically unfavorable. DNA-packaging RNA, pRNA, forms a hexameric complex to serve as a vital component of the DNA translocating motor of bacterial virus Phi29. The sequential action of six pRNA ensures continual function in the DNA translocation process. The Phi29 motor has been assembled with purified components synthesized by chemical or biotechnological approaches and is able to pump the viral DNA into the protein shell in vitro. pRNA dimers are the building blocks of the hexamer. The computer models of the three-dimensional structure of the motor was constructed based on experimental data derived from photoaffinity cross-linking by psoralen, phenphi (cis-Rh(1,10-phenanthroline)(9,10-phenan-threnequinone diimine)Cl(2)(+)), and azidophenacyl; chemical modification and chemical modification interference with dimethyl sulfate, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate, and kethoxal; complementary modification; and nuclease probing by single- and double-stranded specific RNases. The shapes of these computer models are very similar to the published pRNA images of cryo-atomic force microscopy. pRNA hexamer docking with the connector crystal structure reveals a very impressive match with the available biochemical, genetic, and physical data.     

Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging

Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear. It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple of two and three, thus, a hexamer.     

Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.     

High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures.

Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.     

Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.     

A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.     

Boundary of pRNA functional domains and minimum pRNA sequence requirement for specific connector binding and DNA packaging of phage phi29.

Bacteriophage phi29 utilizes a viral-encoded 120-base RNA (pRNA) to accomplish dsDNA packaging into a preformed procapsid. Six pRNAs bind to the procapsid and work sequentially. The pRNA contains two functional domains, one for binding to the DNA translocating connector, and the other for interacting with another component of the DNA packaging machinery during DNA translocation. By UV crosslinking, the pRNA was found to bind to the connector specifically and not to the capsid or scaffolding proteins. When purified connectors were incubated with pRNA, rosette-like connector oligomers were observed. These oligomers were found to contain pRNA. A series of deletion mutants of the pRNA were constructed and their ability to perform various tasks involved in phi29 assembly were assayed. The minimum sizes of the pRNA needed for the following activities have been determined: (1) specific binding to procapsid or to connectors; (2) connector or procapsid binding with full efficiency compared with wild-type pRNA; and (3) genomic DNA packaging. In summary, bases 37-91 (55 nt) comprised the minimum sequence required for specific connector binding, although with lower efficiency; bases 6-113 (105 nt with the additional deletion of two nonessential bases, C109 and A106) comprised the minimum sequence required for full connector binding activity; and bases 1-117 comprised the minimum sequence needed for full DNA packaging activity. These data indicate clearly that the helical region composed of bases 1-6 and 113-117 plays a crucial role in DNA translocation, but is dispensable for connector binding. A model for the role of the pRNA in DNA packaging was also presented.     

Different sequences show similar quaternary interaction stabilities in prohead viral RNA self-assembly

Prohead RNA (pRNA) is an essential component of the self-assembling φ29 bacteriophage DNA packaging motor. Different related species of bacteriophage share only 12% similarity in pRNA sequences. The secondary structure for pRNA is conserved, however. In this study, we present evidence for self-assembly in different pRNA sequences and new measurements of the energetics for the quaternary interactions in pRNA dimers and trimers. The energetics for self-assembly in different pRNA sequences are similar despite very different sequences in the loop-loop interactions. The architecture surrounding the interlocking loops contributes to the stability of the pRNA quaternary interactions, and sequence variation outside the interlocking loops may counterbalance the changes in the loop sequences. Thus, the evolutionary divergence of pRNA sequences maintains not only conservation of function and secondary structure but also stabilities of quaternary interactions. The self-assembly of pRNA can be fine-tuned with variations in magnesium chloride, sodium chloride, temperature, and concentration. The ability to control pRNA self-assembly holds promise for the development of nanoparticle therapeutic applications for this biological molecule. The pRNA system is well suited for future studies to further understand the energetics of RNA tertiary and quaternary interactions, which can provide insight into larger biological assemblies such as viruses and biomolecular motors.     

Effects of peptide dimerization on pore formation: Antiparallel disulfide-dimerized magainin 2 analogue

To elucidate the effects of peptide dimerization on pore formation by magainin 2 (MG2), a covalently linked antiparallel dimer of the MG2 analogue [(F5Y, L6C, F16W, I20C-MG2)(2): II] was synthesized based on the dimer structure revealed by our NMR study. The interactions of the dimer with lipid bilayers were investigated by CD and fluorescence in comparison with a monomer analogue (F5Y, F16W-MG2: I). Similar to I, II was found to form a peptide-lipid supramolecular complex pore accompanied with lipid flip-flop and peptide translocation. The pore formed by II was characterized by a slightly larger pore diameter and a threefold longer lifetime than that of I, although the pore formation rate of the dimer was lower than that of the monomer. The coexistence of the dimer and the monomer exhibited slight but significant synergism in membrane permeabilization, which was maximal at a monomer/dimer ratio of 3. Therefore, we concluded that a pentameric pore composed of one pore-stabilizing dimer and three monomers maximized the overall leakage activity in keeping with our kinetic prediction.     

A comparison of the dynamic behavior of monomeric and dimeric insulin shows structural rearrangements in the active monomer

Molecular dynamics (MD) simulations (5-10ns in length) and normal mode analyses were performed for the monomer and dimer of native porcine insulin in aqueous solution; both starting structures were obtained from an insulin hexamer. Several simulations were done to confirm that the results obtained are meaningful. The insulin dimer is very stable during the simulation and remains very close to the starting X-ray structure; the RMS fluctuations calculated from the MD simulation agree with the experimental B-factors. Correlated motions were found within each of the two monomers; they can be explained by persistent non-bonded interactions and disulfide bridges. The correlated motions between residues B24 and B26 of the two monomers are due to non-bonded interactions between the side-chains and backbone atoms. For the isolated monomer in solution, the A chain and the helix of the B chain are found to be stable during 5ns and 10ns MD simulations. However, the N-terminal and the C-terminal parts of the B chain are very flexible. The C-terminal part of the B chain moves away from the X-ray conformation after 0.5-2.5ns and exposes the N-terminal residues of the A chain that are thought to be important for the binding of insulin to its receptor. Our results thus support the hypothesis that, when monomeric insulin is released from the hexamer (or the dimer in our study), the C-terminal end of the monomer (residues B25-B30) is rearranged to allow binding to the insulin receptor. The greater flexibility of the C-terminal part of the beta chain in the B24 (Phe-->Gly) mutant is in accord with the NMR results. The details of the backbone and side-chain motions are presented. The transition between the starting conformation and the more dynamic structure of the monomers is characterized by displacements of the backbone of Phe B25 and Tyr B26; of these, Phe B25 has been implicated in insulin activation.     

Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan-binding protein 1 (HABP1)/p32/gC1qR.

Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human fibroblast exists in different oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modification of Cys186. The gradual structural transition caused by cysteine-mediated disulfide linkage is evident as the fluorescence intensity increases with increasing Hg(2+) concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the affinity of the hexamer for different ligands. The hexamer shows enhanced affinity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function.     

Virulence factor of potato virus Y, genome-attached terminal protein VPg, is a highly disordered protein

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5' end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.