Structure of a malaria parasite antigenic determinant displayed on filamentous bacteriophage determined by NMR spectroscopy: implications for the structure of continuous peptide epitopes of proteins

The NANP repeating sequence of the circumsporozoite protein of Plasmodium falciparum was displayed on the surface of fd filamentous bacteriophage as a 12-residue insert (NANP)(3) in the N-terminal region of the major coat protein (pVIII). The structure of the epitope determined by multidimensional solution NMR spectroscopy of the modified pVIII protein in lipid micelles was shown to be a twofold repeat of an extended and non-hydrogen-bonded loop based on the sequence NPNA, demonstrating that the repeating sequence is NPNA, not NANP. Further, high resolution solid-state NMR spectra of intact hybrid virions containing the modified pVIII proteins demonstrate that the peptides displayed on the surface of the virion adopt a single, stable conformation; this is consistent with their pronounced immunogenicity as well as their ability to mimic the antigenicity of their native parent proteins.     

De novo folding of membrane proteins: an exploration of the structure and NMR properties of the fd coat protein

De novo folding simulations of the major pVIII coat protein from filamentous fd bacteriophage, using a newly developed implicit membrane generalized Born model and replica-exchange molecular dynamics, are presented and discussed. The quality of the predicted structures, judged by comparison of the root-mean-square deviations of a room temperature ensemble of conformations from the replica-exchange simulations and experimental structures from both solid-state NMR in lipid bilayers and solution-phase NMR on the protein in micelles, was quite good, reinforcing the general quality of the folding simulations. The transmembrane helical segment of the protein was well defined in comparison with experiment and the amphipathic helical fragment remained at the membrane/aqueous phase boundary while undergoing significant conformational flexibility due to the loop connecting the two helical segments of the protein. Additional comparisons of computed solid-state NMR properties, the 15N chemical shift and 15N-1H dipolar coupling constants, showed semi-quantitative agreement with the corresponding measurements. These findings suggest an emerging potential for the de novo investigation of integral membrane peptides and proteins and a mechanism to assist experimental approaches to the characterization and structure determination of these important systems.     

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.     

Rotational motion of cytochrome c oxidase in phospholipid vesicles

Natural abundance ¹³C and high field ¹H NMR spectroscopy are used to characterize the major coat protein of the filamentous bacteriophage fd in sodium dodecyl sulfate micelles. Chemical shift dispersion of protein resonances, slow and differential exchange rates of amide protons, and relaxation parameters of the alpha carbons of the protein indicate that the detergent solubilized coat protein has a stable native conformation. The structure of the coat protein in micelles differs from that found for typical globular proteins in solution in that parts of the peptide backbone exhibit rapid segmental motion.     

Enhanced Sampling of Coarse-Grained Transmembrane-Peptide Structure Formation from Hydrogen-Bond Replica Exchange

Protein structure formation in the membrane highlights a grand challenge of sampling in computer simulations, because kinetic traps and slow dynamics make it difficult to find the native state. Exploiting increased fluctuations at higher temperatures can help overcome free-energy barriers, provided the membrane’s structure remains stable. In this work, we apply Hamiltonian replica-exchange molecular dynamics, where we only tune the backbone hydrogen-bond strength to help reduce the propensity of long-lived misfolded states. Using a recently developed coarse-grained model, we illustrate the robustness of the method by folding different WALP transmembrane helical peptides starting from stretched, unstructured conformations. We show the efficiency of the method by comparing to simulations without enhanced sampling, achieving folding in one example after significantly longer simulation times. Analysis of the bilayer structure during folding provides insight into the local membrane deformation during helix formation as a function of chain length (from 16 to 23 residues). Finally, we apply our method to fold the 50-residue-long major pVIII coat protein (fd coat) of the filamentous fd bacteriophage. Our results agree well with experimental structures and atomistic simulations based on implicit membrane models, suggesting that our explicit CG folding protocol can serve as a starting point for better-refined atomistic simulations in a multiscale framework.     

The structure of a filamentous bacteriophage

Many thin helical polymers, including bacterial pili and filamentous bacteriophage, have been seen as refractory to high-resolution studies by electron microscopy. Studies of the quaternary structure of such filaments have depended upon techniques such as modeling or X-ray fiber diffraction, given that direct visualization of the subunit organization has not been possible. We report the first image reconstruction of a filamentous virus, bacteriophage fd, by cryoelectron microscopy. Although these thin ( approximately 70 A in diameter) rather featureless filaments scatter weakly, we have been able to achieve a nominal resolution of approximately 8 A using an iterative helical reconstruction procedure. We show that two different conformations of the virus exist, and that in both states the subunits are packed differently than in conflicting models previously proposed on the basis of X-ray fiber diffraction or solid-state NMR studies. A significant fraction of the population of wild-type fd is either disordered or in multiple conformational states, while in the presence of the Y21M mutation, this heterogeneity is greatly reduced, consistent with previous observations. These results show that new computational approaches to helical reconstruction can greatly extend the ability to visualize heterogeneous protein polymers at a reasonably high resolution.     

On the structures of filamentous bacteriophage Ff (fd,f1, M13)

The filamentous bacteriophage (Inovirus) strain Ff (fd, f1, M13) is widely used in molecular biophysics as a simple model system. A low resolution molecular model of the fd protein coat has been reported, derived from iterative helical real space reconstruction of cryo-electron micrographs (cryoEM). This model is significantly different from the model previously derived from X-ray fibre diffraction and solid-state NMR. We show that the cryoEM model agrees neither with solid-state NMR data nor with X-ray fibre diffraction data of fd, and has some puzzling structural features, for instance nanometre holes through the protein coat. We refine the cryoEM model against the X-ray data, and find that the model after refinement closely approximates the model derived directly from X-ray fibre diffraction and solid-state NMR data. We suggest possible reasons for the differences between the models derived from cryoEM and X-ray diffraction.     

Variable electrostatic interaction between DNA and coat protein in filamentous bacteriophage assembly

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the DNA of the class I filamentous bacteriophage fd, which infects Escherichia coli. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter, generating plasmid pKf8P. Bacteriophage fd gene VIII was similarly cloned into the plasmid pEMBL9+, enabling it to be subjected to site-directed mutagenesis. By this means the positively charged lysine residue at position 48, one of four positively charged residues near the C terminus of the protein, was turned into a negatively charged glutamic acid residue. The mutated fd gene VIII was cloned back from the pEMBL plasmid into the expression plasmid pKK223-3, creating plasmid pKE48. In the presence of the inducer isopropyl-beta-D-thiogalactoside, the wild-type and mutated coat protein genes were strongly expressed in E. coli TG1 cells transformed with plasmids pKf8P and pKE48, respectively, and the product procoat proteins underwent processing and insertion into the E. coli cell inner membrane. A net positive charge of only 2 on the side-chains in the C-terminal region is evidently sufficient for this initial stage of the virus assembly process. However, the mutated coat protein could not encapsidate the DNA of bacteriophage R252, an fd bacteriophage carrying an amber mutation in its own gene VIII, when tested on non-suppressor strains of E. coli. On the other hand, elongated hybrid bacteriophage particles could be generated whose capsids contained mixtures of wild-type (K48) and mutant (E48) subunits. This suggests that the defect in assembly may occur at the initiation rather than the elongation step(s) in virus assembly. Other mutations of lysine-48 that removed or reversed the positive charge at this position in the C-terminal region of the coat protein were also found to lead to the production of commensurately longer bacteriophage particles. Taken together, these results indicate direct electrostatic interaction between the DNA and the coat protein in the capsid and support a model of non-specific binding between DNA and coat protein subunits with a stoicheiometry that can be varied during assembly.     

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.     

Cobalt ion mediated self-assembly of genetically engineered bacteriophage for biomimetic Co-Pt hybrid material

Biological scaffolds are used for the synthesis of inorganic materials due to their ability to self-assemble and nucleate crystal formation. We report the self-assembly of engineered M13 bacteriophage as a template for Co-Pt crystals. A M13 phage library with an octapeptide library on the major coat protein (pVIII) was used for selection of binders to cobalt ions. Fibrous structures with directionally ordered M13 phage were obtained by interaction with cobalt ions. Co-Pt alloys were synthesized on the fibrous scaffold, and their magnetic properties were characterized. The mineralization showed organized nanoparticles on fibrous bundles. This approach using the phage pVIII library allows for genetic selection that both induces assembly of the phage and directs mineralization of the selected inorganic material.     

Chemical modification of the coat protein in bacteriophage fd and orientation of the virion during assembly and disassembly.

The major (gene VIII) coat protein of bacteriophage fd was radiolabelled by treating the virus with methyl[3H]acetimidate without causing any loss of infectivity. Complete amidination of lysine-8 in the amino acid sequence of the protein was achieved but little or no modification of the lysine residues near the C terminus was observed. This supports the assumption that the coat protein is oriented in the viral filament with its N terminus on the outside and its C-terminal region abutting the DNA. Escherichia coli was co-infected with radiolabelled bacteriophage and with unlabelled miniphage, a shorter defective form of phage fd. Radiolabel was detected in the progeny miniphage, proving that individual coat protein subunits can be recycled and assembled onto progeny miniphage DNA. About 35% of the coat protein subunits of phage particles infecting E. coli were recycled in 1 h. These facts support a model of the assembly and disassembly of the virion at the bacterial membrane in which the end of the particle containing the minor adsorption (gene III) protein, which is presumably the first to disassemble during infection, is the last to assemble during morphogenesis.     

New vectors for peptide display on the surface of filamentous bacteriophage

We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion. These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits.     

Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage

Background

Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display.

Methodology/Principal Findings

Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient.

Conclusions/Significance

Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.     

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.     

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.     

Orientations of tyrosines 21 and 24 in coat subunits of Ff filamentous virus: determination by Raman linear intensity difference spectroscopy and implications for subunit packing.

Virions of the Ff group of bacteriophages (fd, f1, M13) are morphologically identical filaments (approximately 6-nm diameter x approximately 880-nm length) in which a covalently closed, single-stranded DNA genome is sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit (pVIII). Orientations of pVIII tyrosines (Tyr21 and Tyr24) with respect to the filament axis have been determined by Raman linear intensity difference (RLID) spectroscopy of flow-oriented mutant virions in which the tyrosines were independently mutated to methionine. The results show that the twofold axis of the phenolic ring (C1-C4 line) of Tyr21 is inclined at 39.5 +/- 1.4 degrees from the virion axis, and that of Tyr24 is inclined at 43.7 +/- 0.6 degrees. The orientation determined for the Tyr21 phenol ring is close to that of a structural model previously proposed on the basis of fiber x-ray diffraction results (Protein Data Bank, identification code 1IFJ). On the other hand, the orientation determined for the Tyr24 phenol ring differs from the diffraction-based model by a 40 degrees rotation about the Calpha-Cbeta bond. The RLID results also indicate that each tyrosine mutation does not greatly affect the orientation of either the remaining tyrosine or single tryptophan (Trp26) of pVIII. On the basis of these results, a refined model is proposed for the coat protein structure in Ff.