Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.     

The protein capsid of filamentous bacteriophage PH75 from Thermus thermophilus

The PH75 strain of filamentous bacteriophage (Inovirus) grows in the thermophilic bacterium Thermus thermophilus at 70 degrees C. We have characterized the viral DNA and determined the amino acid sequence of the major coat protein, p8. The p8 protein is synthesized without a leader sequence, like that of bacteriophage Pf3 but unlike that of bacteriophage Pf1, both of which grow in the mesophile Pseudomonas aeruginosa. X-ray diffraction patterns from ordered fibres of the PH75 virion are similar to those from bacteriophages Pf1 and Pf3, indicating that the protein capsid of the PH75 virion has the same helix symmetry and subunit shape, even though the primary structures of the major coat proteins are quite different and the virions assemble at very different temperatures. We have used this information to build a molecular model of the PH75 protein capsid based on that of Pf1, and refined the model by simulated annealing, using fibre diffraction data extending to 2.4 A resolution in the meridional direction and to 3.1 A resolution in the equatorial direction. The common design may reflect a fundamental motif of alpha-helix packing, although differences exist in the DNA packaging and in the means of insertion of the major coat protein of these filamentous bacteriophages into the membrane of the host bacterial cell. These may reflect differences in the assembly mechanisms of the virions.     

Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state ¹H/¹⁵N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.     

Comparison of protein and deoxyribonucleic acid backbone structures in fd and Pf1 bacteriophages

The conformations of the protein and nucleic acid backbones in the filamentous viruses fd and Pf1 are characterized by one- and two-dimensional solid-state NMR experiments on oriented virus solutions. Striking differences are observed between fd and Pf1 in both their protein and DNA structures. The coat proteins of fd and Pf1 are almost entirely alpha helical and in both viruses most of the helix is oriented parallel to the filament axis. fd coat protein is one stretch of alpha helix that is slightly slued about the filament axis. In Pf1 coat protein two distinct sections of alpha helix are present, the smaller of which is tilted with respect to the filament axis by about 20 degrees. The DNA backbone structure of fd is completely disordered. By contrast, the DNA backbone of Pf1 is uniformly oriented such that all of the phosphodiester groups have the O-P-O plane of the nonesterified oxygens approximately perpendicular to the filament axis.     

Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.     

Intensity of the polarized Raman band at 1340-1345 cm-1 as an indicator of protein alpha-helix orientation: application to Pf1 filamentous virus

Raman spectra of oriented alpha-helical protein molecules exhibit a prominent band near 1340-1345 cm(-)(1), the intensity of which is highly sensitive to molecular orientation. Polarization of the 1340-1345 cm(-)(1) marker is evident in Raman spectra of alpha-helical poly-L-alanine (alphaPLA) and alpha-helical poly-gamma-benzyl-L-glutamate (alphaPBLG). Corresponding polarization is also observed in Raman spectra of the filamentous virus Pf1, which is an assembly of alpha-helical coat protein molecules. In alphaPLA and alphaPBLG, we assign the band to a normal mode of symmetry type E(2) and specifically to a vibration localized in the (O=C)-C(alpha)-H linkages of the main chain peptide group. Although strict helical symmetry does not apply to coat subunits of filamentous viruses, an approximate E(2)-type mode may be presumed to account for a corresponding Raman band of Pf1 and fd filamentous viruses. Spectroscopic studies of N-methylacetamide and isotopically-edited fd viruses support the present assignment of the 1340-1345 cm(-)(1) band. Polarization anisotropy indicates that this band may be exploited as a novel indicator of protein alpha-helix orientation. Application of this approach to the polarized Raman spectrum of Pf1 suggests that, on average, the axis of the alpha-helical coat protein subunit in the native virion structure forms an angle of 20 +/- 10 degrees with respect to the virion axis.     

Structure and dynamics of the Pf1 filamentous bacteriophage coat protein in micelles

The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation in detergent micelles. High-resolution 1H and 15N NMR experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pf1 coat protein in sodium dodecyl sulfate micelles. Uniform and specific-site 15N labels enable the immobile backbone sites to be identified by their 1H/15N heteronuclear nuclear Overhauser effect and allow the assignment of 1H and 15N resonances. About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments. The combination of results from 1H/15N heteronuclear correlation, 1H homonuclear correlation, and 1H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure.     

Nuclear magnetic resonance of the filamentous bacteriophage fd.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.     

Line shape analyses for water 17O NMR quintet observed in a bacteriophage Pf1 solution at different temperatures

Partially resolved 17O NMR quintet was observed in a filamentous bacteriophage Pf1 solution at 70 degrees C with a quadrupole splitting approximately 100 Hz. As the temperature decreased, the resolution was reduced but the line shapes were still indicative of residual quadrupole splitting. Line shape analyses were performed using the quadrupolar relaxation theory for spin 5/2. The contribution to the residual quadrupole splitting from the electric field gradients stemming from the phage filaments, which were oriented in the magnet, was taken into account. As a result, the observed 17O spectra at different temperatures were simulated and the hydration number of the phage DNA was determined.     

Neutron diffraction studies of the structure of filamentous bacteriophage Pf1. Demonstration that the coat protein consists of a pair of alpha-helices with an intervening, non-helical surface loop

The structure of filamentous bacteriophage Pf1 has been studied using neutron diffraction from magnetically oriented gels of native and specifically deuterated phage. These methods have been used to determine the positions of the two methionine, two tyrosine and six isoleucine residues of the coat protein. Combined with the positions of the five valine residues previously determined, they represent one third (15 of 46) of the residues of the coat protein. These 15 amino acid residue positions have been used as the basis for constructing a model for the protein consisting of two alpha-helices with an intervening surface loop. The first helix extends from near the amino terminus to Ile12. The second helix extends from Lys20 to at least Met42, and may contain a bend between Ile32 and Val35. The two helices are tilted by about 15 degrees relative to one another, and are positioned in such a way that they appear to be bound end-to-end by main-chain hydrogen bonds. The intervening, non-helical loop, made up of Thr13 to Met19, connects the two helices without disrupting the pattern of main-chain hydrogen bonding, but does not result in a bend in the otherwise continuous helical structure. This model is used to predict the approximate positions of all amino acid residues in the Pf1 protein coat, providing a basis for further understanding of a number of viral properties including the symmetry transitions, the non-isomorphism of heavy-atom derivatives, and the protein-protein and protein-DNA interactions in the virion.     

Consensus structure of Pf1 filamentous bacteriophage from X-ray fibre diffraction and solid-state NMR

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.     

Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Magic angle spinning solid-state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10 °C. Comparisons of NMR spectra recorded above and below 10 °C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side chains of neighboring subunits. These contacts are important constraints for structure modeling.     

Structural details of the thermophilic filamentous bacteriophage PH75 determined by polarized Raman microspectroscopy

The filamentous virus PH75, which infects the thermophile Thermus thermophilus, consists of a closed DNA strand of 6500 nucleotides encapsidated by 2700 copies of a 46-residue coat subunit (pVIII). The PH75 virion is similar in composition to filamentous viruses infecting mesophilic bacteria but is distinguished by in vivo assembly at 70 degrees C and thermostability to at least 90 degrees C. Structural details of the PH75 assembly are not known, although a fiber X-ray diffraction based model suggests that capsid subunits are highly alpha-helical and organized with the same symmetry (class II) as in the mesophilic filamentous phages Pf1 and Pf3 [Pederson et al. (2001) J. Mol. Biol. 309, 401-421]. This is distinct from the symmetry (class I) of phages fd and M13. We have employed polarized Raman microspectroscopy to obtain further details of PH75 architecture. The spectra are interpreted in combination with known Raman tensors for modes of the pVIII main chain (amide I) and Trp and Tyr side chains to reveal the following structural features of PH75: (i) The average pVIII peptide group is oriented with greater displacement from the virion axis than peptide groups of fd, Pf1, or Pf3. The data correspond to an average helix tilt angle of 25 degrees in PH75 vs 16 degrees in fd, Pf1, and Pf3. (ii) The indolyl ring of Trp 37 in PH75 projects nearly equatorially from the subunit alpha-helix axis, in contrast to the more axial orientations for Trp 26 of fd and Trp 38 of Pf3. (iii) The phenolic rings of Tyr 15 and Tyr 39 project along the subunit helix axis, and one phenoxyl engages in hydrogen-bonding interaction that has no counterpart in either fd or Pf1 tyrosines. Also, in contrast to fd, Pf1, and Pf3, the packaged DNA genome of PH75 exhibits no Raman anisotropy, suggesting that DNA bases are not oriented unidirectionally within the nucleocapsid assembly. The structural findings are discussed in relation to intrasubunit and intersubunit interactions that may confer hyperthermostability to the PH75 virion. A refined molecular model is proposed for the PH75 capsid subunit.     

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Shuffling of structural elements in filamentous bacteriophages

All class II filamentous bacteriophage coat proteins contain a conserved, 12-amino acid sequence highly homologous to the loop portion of the EF-hand Ca²⁺-binding motif. The Pf3 coat protein contains two regions of homology to this sequence. The 12-amino acid sequence corresponds to a region of the Pf1 coat protein whose structure is controversial. In some models of the virus structure, this region is α-helical. In others, it forms a loop that folds back on itself. The similarity of this region to the loop in the helix-loop-helix Ca²⁺-binding motif suggests that it takes on a loop structure in the virion. Each filamentous phage lacks at least one residue normally involved in Ca²⁺-coordination, consistent with the relatively weak Ca²⁺ binding properties of the filamentous phages. Consideration of the structure of the coat protein in the membrane and in the virus particle indicates that the protein may be more effective in binding cations in its membrane-bound form than in the virus particle. This suggests that release of cations from this loop may be an obligate step during assembly of the proteins into the virus particle. Proteins 27:405–409, 1997. © 1997 Wiley-Liss, Inc.     

Structure similarity, difference and variability in the filamentous viruses fd, If1, IKe, Pf1 and Xf. Investigation by laser Raman spectroscopy

The filamentous bacteriophages fd, If1, IKe, Pf1, Xf and Pf3 in aqueous solutions of low, moderate and high ionic strength have been investigated as a function of temperature by laser Raman difference spectroscopy. By analogy with Raman spectra of model compounds and viruses of known structure, the data reveal the following structural features: the predominant secondary structure of the coat protein subunit in each virus is the alpha-helix, but the amount of alpha-helix differs from one virus to another, ranging from an estimated high of 100% in Pf1 to a low of approximately 50% in Xf. The molecular environment and intermolecular interactions of tyrosine, tryptophan and phenylalanine residues differ among the different viruses, as do the conformations of aliphatic amino acid side-chains. The foregoing features of coat protein structure are highly sensitive to changes in Na+ concentration, temperature or both. The backbones of A-DNA and B-DNA structures do not occur in any of the viruses, and unusual DNA structures are indicated for all six viruses. The alpha-helical protein subunits of Pf1, like those of Pf3 and Xf, can undergo reversible transitions to beta-sheet structures while retaining their association with DNA; yet fd, IKe and If1 do not undergo such transitions. Raman intensity changes with ionic strength or temperature suggest that transgauche rotations of aliphatic amino acid side-chains and stacking of aromatic side-chains are important structural variables in each virus.     

Structural characterization of the filamentous bacteriophage PH75 from Thermus thermophilus by Raman and UV-resonance Raman spectroscopy

The filamentous bacteriophage PH75, which infects the thermophile T. thermophilus, assembles in vivo at 70 degrees C and is stable to at least 90 degrees C. Although a high-resolution structure of PH75 is not available, the virion is known to comprise a closed single-stranded (ss) DNA circle of 6500 nucleotides sheathed by a capsid comprising 2700 copies of a 46-residue subunit (pVIII). Here, we employ Raman and UV-resonance Raman (UVRR) spectroscopy to identify structural details of the pVIII and DNA constituents of PH75 that may be related to the high thermostability of the native virion assembly. Analysis of the Raman amide I and amide III signatures reveals that the capsid subunit secondary structure is predominantly (87%) alpha-helical but contains a significant number of residues (6 +/- 1 or 13 +/- 3%) differing from the canonical alpha-helix. This minor structural component is not apparent in capsid subunits of the mesophilic filamentous phages, fd, Pf1, and Pf3, previously examined at similar spectral resolution. The Raman signature of PH75 also differs from those of fd, Pf1, and Pf3 by virtue of an unusual alanine marker (898 cm(-)(1) band), which is attributed to C(alpha)-H hydrogen-bond donation by subunit Ala residues. Because alanines of the PH75 subunit occur primarily within sXXXs motifs (where s is a small side chain, e.g. Gly, Ala, Ser), and because the occurrence of such motifs in alpha-helices is believed to thermostabilize interhelix associations via C(alpha)-H...O interactions [G. Kleiger et al. (2002) Biochemistry 41, 5990-5997], we propose that such hydrogen bonding may explain both the alanyl and amide I/III markers of PH75 capsid subunits and that C(alpha)-H...O interactions may serve as a significant source of virion thermostabilization. Raman and UVRR signatures of PH75 are also distinguished from those of fd, Pf1, and Pf3 by several marker bands that are indicative of hydrophilic Trp and Tyr environments, including hydrogen bonding interactions of aromatic ring substituents. These interactions are likewise proposed as contributors to the high thermostability of PH75 vis-a-vis fd, Pf1, and Pf3. Finally, PH75 is the only filamentous phage exhibiting UVRR markers diagnostic of a highly base-stacked ssDNA genome incorporating the low energy C2'-endo/anti deoxynucleoside conformation. The present results suggest that both intersubunit interactions and genome organization contribute to the enhanced thermostability of PH75 relative to mesophilic filamentous bacteriophages.