Protein-lipid interactions of bacteriophage M13 gene 9 minor coat protein (Review)

Gene 9 protein is one of the minor coat proteins of bacteriophage M13. The protein plays a role in the assembly process by associating with the host membrane by protein-lipid interactions. The availability of chemically synthesized protein has enabled the biophysical characterization of the membrane-bound state of the protein by using model membrane systems. This paper summarizes, discusses and further interprets this work in the light of the current state of the literature, leading to new possible models of the coat protein in a membrane. The biological implications of these findings related to the membrane-bound phage assembly are indicated.     

The in situ aggregational and conformational state of the major coat protein of bacteriophage M13 in phospholipid bilayers mimicking the inner membrane of host Escherichia coli

The major coat protein of bacteriophage M13 has been reconstituted into phospholipids with a composition comparable to that found in the host (Escherichia coli) inner membrane. Reconstitution experiments have revealed conditions in which the alpha-oligomeric state is favored over the beta-polymeric state. Discrimination between the two states of the membrane-bound coat protein (alpha-oligomeric and beta-polymeric states) has been achieved using high-performance size-exclusion chromatography and circular dichroism. Interprotein electrostatic interactions, probably induced by head-tail binding, are initiated and facilitating the aggregation-related conformational change process, in which alpha-oligomeric coat protein is converted into beta-polymeric coat protein. A model for this beta-polymerization process of the coat protein is presented. The alpha-helical protein has been studied by the in situ Trp fluorescence quantum yield. This shows that the average distances between coat proteins decrease upon lowering the L/P ratio. In situ cross-linking reactions of the coat protein at high L/P ratios reveal a monomeric state, thus excluding specific aggregation of the coat protein. A monomeric state of detergent-solubilized coat protein is also observed using SDS-PAGE and SDS-HPSEC. On the basis of these results, the smallest in situ aggregational entity of the coat protein is proposed to be a monomer. This finding is discussed in relation to the functional state of the M13 coat protein in the membrane-bound assembly and disassembly processes during infection.     

Polyoma large T antigen regulates the integration of viral DNA sequences into the genome of transformed cells

The major coat protein of coliphage M13 is an integral protein of the E. coli plasma membrane prior to its assembly into new virus particles. It is generated from its precursor, procoat, by a membrane-bound leader peptidase. We now describe the reconstitution of a highly purified preparation of this enzyme into vesicles of E. coli phospholipids. These vesicles bind procoat made in vitro and procoat isolated from in vitro synthesis. Both the crude and the purified substrates were converted posttranslationally to coat protein. A significant proportion of the coat protein becomes inserted into the vesicle bilayer, with the N terminus facing the vesicle interior and the C terminus exposed to the external medium. These results strongly suggest that highly purified leader peptidase from E. coli and phospholipids are the only components necessary to mediate the binding, processing and insertion of this integral membrane protein.     

Membrane assembly of M13 major coat protein: evidence for a structural adaptation in the hinge region and a tilted transmembrane domain

New insights into the low-resolution structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process.     

Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in model membrane systems, which varied in lipid head group and lipid acyl chain composition. By using FTIR spectroscopy and subsequent band analysis a quantitative analysis of the secondary structure of the protein was obtained. The secondary structure of the gene 9 protein predominantly consists of alpha-helical (67%) and turn (33%) structures. The turn structure is likely to be located C-terminally where it has a function in recognizing the phage DNA during bacteriophage assembly. Attenuated total reflection FTIR spectroscopy was used to determine the orientation of gene 9 protein in the membrane, revealing that the alpha-helical domain is mainly transmembrane. The conformational and orientational measurements result in two models for the gene 9 protein in the membrane: a single transmembrane helix model and a two-helix model consisting of a 15 amino acid long transmembrane helix and a 10 amino acid long helix oriented parallel to the membrane plane. Potential structural consequences for both models are discussed.     

Membrane assembly of the bacteriophage Pf3 major coat protein

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.     

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.     

Both hydrophobic domains of M13 procoat are required to initiate membrane insertion.

M13 procoat protein has two hydrophobic domains, one in the leader peptide and one which anchors the mature coat protein in the membrane. Disruption of the membrane anchor region by insertion of arginyl residues does not yield periplasmic coat protein. Instead, the rate of membrane assembly is slowed greater than 100-fold (t1/2 less than 5 s for wild-type, t1/2 greater than 10 min for mutant). The hydrophobic region of mature coat protein not only functions as a membrane anchor, but has an important role in the membrane assembly process per se.     

COPII coat assembly and selective export from the endoplasmic reticulum

The coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). Recent structural and biochemical studies have suggested that the COPII coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the ER membrane that drives the transport vesicle formation. The COPII-coated vesicle formation at the ER membrane is triggered by the activation of the Ras-like small GTPase Sar1 by GDP/GTP exchange, and activated Sar1 in turn promotes COPII coat assembly. Subsequent GTP hydrolysis by Sar1 leads to disassembly of the coat proteins, which are then recycled for additional rounds of vesicle formation. Thus, the Sar1 GTPase cycle is thought to regulate COPII coat assembly and disassembly. Emerging evidence suggests that the cargo proteins modulate the Sar1 GTP hydrolysis to coordinate coat assembly with cargo selection. Here, I discuss the possible roles of the GTP hydrolysis by Sar1 in COPII coat assembly and selective uptake of cargo proteins into transport vesicles.     

Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13

During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.     

Recombinant forms of M13 procoat with an OmpA leader sequence or a large carboxy-terminal extension retain their independence of secY function.

The assembly of phage M13 procoat protein into the plasma membrane of Escherichia coli is independent of the secY protein. To test whether this is caused by the unusually small size of procoat, we fused DNA encoding 103 amino acids to the carboxy-terminal end of the procoat gene. The resulting fusion protein, which attains the same membrane-spanning conformation as mature coat protein, still does not require the secY function for membrane assembly. To determine whether the leader sequence governs interaction with the secY protein, we genetically exchanged the leader peptides between procoat and pro-OmpA, a protein which does require secY for its membrane assembly. Each of the resulting hybrid proteins assembles across the plasma membrane, though at a reduced rate. Membrane assembly of the fusion of procoat leader and OmpA required secY function, whereas assembly of the pro-OmpA leader/coat protein fusion was independent of secY. Properties of the entire procoat molecule, rather than its small size or a specific property of its leader peptide determines its mode of membrane assembly.     

Membrane-bound conformation of M13 major coat protein: a structure validation through FRET-derived constraints

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or F?rster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight alpha-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.     

Common principles in clathrin-mediated sorting at the Golgi and the plasma membrane

Clathrin-mediated vesicular trafficking events underpin the vectorial transfer of macromolecules between several eukaryotic membrane-bound compartments. Classical models for coat operation, focused principally on interactions between clathrin, the heterotetrameric adaptor complexes, and cargo molecules, fail to account for the full complexity of the coat assembly and sorting process. New data reveal that targeting of clathrin adaptor complexes is generally supported by phosphoinositides, that cargo recognition by heterotetrameric adaptors depends on phosphorylation-driven conformational alterations, and that dedicated clathrin-associated sorting proteins (CLASPs) exist to promote the selective trafficking of specific categories of cargo. A host of accessory factors also participate in coat polymerization events, and the independently folded appendage domains that project off the heterotetrameric adaptor core function as recruitment platforms that appear to oversee assembly operations. It is also now clear that focal polymerization of branched actin microfilaments contributes to clathrin-coated vesicle assembly and movement at both plasma membrane and Golgi sites. This improved appreciation of the complex mechanisms governing clathrin-dependent sorting events reveals several common principles of clathrin operation at the Golgi and the plasma membrane.     

Comparison of the dynamics of the membrane-bound form of fd coat protein in micelles and in bilayers by solution and solid-state nitrogen-15 nuclear magnetic resonance spectroscopy

Solid-state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane-bound form of the protein. The residues in the N- and C-terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C-terminal region of the coat protein in micelles; at 25 degrees C only the last two residues are mobile on the 10(9)-Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C-terminal residues are immobile in the virus particles, the mobility of these residues in the membrane-bound form of the protein may be important for the formation of protein-DNA interactions in the assembly process.     

Clathrin: anatomy of a coat protein.

Clathrin is a vesicle coat protein involved in the assembly of membrane and cargo into transport vesicles at the plasma membrane and on certain intracellular organelles. Recently, crystal structures of two separate parts of the clathrin heavy chain, a fragment of the proximal leg and the N-terminal domain, have been analysed, providing the first high-resolution data for a vesicle coat protein. Viewing these structures in the context of a hexagonal barrel coat, recently determined to 21 A by cryo-electron microscopy, provides new insights into the assembly of clathrin coats.     

Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage

The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions.     

DNA packaging orders the membrane of bacteriophage PRD1.

Bacteriophage PRD1 contains a linear dsDNA genome enclosed by a lipid membrane lying within a protein coat. Determination of the structure of the detergent-treated particle to 2 nm by cryo-electron microscopy and three-dimensional reconstruction has defined the position of the major coat protein P3. The coat contains 240 copies of trimeric P3 packed into positions of local 6-fold symmetry on a T = 25 lattice. The three-dimensional structures of the PRD1 virion and a DNA packaging mutant to a resolution of 2.8 nm have revealed specific interactions between the coat and the underlying membrane. The membrane is clearly visible as two leaflets separated by 2 nm and spanned by transmembrane density. The size of the coat does not change upon DNA packaging. Instead, the number of interactions seen between the protein shell and the membrane and the order of the membrane components increase. Thus the membrane of PRD1 plays a role in assembly which is akin to that played by the nucleocapsid in other membrane viruses.     

A second-site suppressor of a folding defect functions via interactions with a chaperone network to improve folding and assembly in vivo

Single amino acid substitutions in a protein can cause misfolding and aggregation to occur. Protein misfolding can be rescued by second-site amino acid substitutions called suppressor substitutions (su), commonly through stabilizing the native state of the protein or by increasing the rate of folding. Here we report evidence that su substitutions that rescue bacteriophage P22 temperature-sensitive-folding (tsf) coat protein variants function in a novel way. The ability of tsf:su coat proteins to fold and assemble under a variety of cellular conditions was determined by monitoring levels of phage production. The tsf:su coat proteins were found to more effectively utilize P22 scaffolding protein, an assembly chaperone, as compared with their tsf parents. Phage-infected cells were radioactively labelled to quantify the associations between coat protein variants and folding and assembly chaperones. Phage carrying the tsf:su coat proteins induced more GroEL and GroES, and increased formation of protein:chaperone complexes as compared with their tsf parents. We propose that the su substitutions result in coat proteins that are more assembly competent in vivo because of a chaperone-driven kinetic partitioning between aggregation-prone intermediates and the final assembled state. Through more proficient use of this chaperone network, the su substitutions exhibit a novel means of suppression of a folding defect.     

Filamentous Bacterial viruses

The filamentous bacterial virus is a simple and well-characterized model system for studying how genetic information is transformed into molecular machines. The viral DNA is a single-stranded circle coding for about 10 proteins. The major viral coat protein is largely α-helical, with about 46 amino acid residues. Several thousand identical copies of this protein in a helical array form a hollow cylindrical tube 1–2μ long, of outer diameter 60 Å and inner diameter 20 Å, with the twisted circular DNA extending down the core of the tube. Before assembly, the viral coat protein spans the cell membrane, and assembly involves extrusion of the coat from the membrane. X-ray fibre diffraction patterns of the Pf 1 species of virus at 4°C, oriented in a strong magnetic field, give three-dimensional data to 4 Å resolution. An electron density map calculated from native virus and a single iodine derivative, using the maximum entropy technique, shows a helix pitch of 5.9 Å. This may indicate a stretched A-helix, or it may indicate a partially 3₁₀ helix conformation, resulting from the fact that the coat protein is an integral membrane protein before assembly, and is still in the hydrophobic environment of other coat proteins after assembly.