Characterization of a Dual-Function Domain That Mediates Membrane Insertion and Excision of Ff Filamentous Bacteriophage

The filamentous phage Ff (f1, fd, or M13) of Escherichia coli is assembled at the cell membranes by a process that is morphologically similar to that of pilus assembly. The release of the filament virion is mediated by excision from the membrane; conversely, entry into a host cell is mediated by insertion of the virion coat proteins into the membrane. The N-terminal domains of the minor virion protein pIII have the sole role of binding to host receptors during infection. In contrast, the C domain of pIII is required for two opposite functions: insertion of the virion into the membrane during infection and excision at the termination step of assembly/secretion. We identified a 28-residue-long segment in the pIII C domain, which is required for phage entry but dispensable for release from the membrane at the end of assembly. This segment, which we named the infection-competence segment (ICS), works only in cis with the N-terminal receptor-binding domains and does not require the equivalent ICS sequences in other subunits within the virion cap. The ICS contains a predicted amphipathic α-helix and is rich in small amino acids, Gly, Ala, and Ser, which are arranged as a [small]XXX[small]XX[small]XXX[small]XXX[small] motif. Scanning Ala/Gly mutagenesis of ICS showed that small residues are compatible with infection. Overall, organization of the C domain is reminiscent of α-helical pore-forming toxins' membrane insertion domains. The unique ability of pIII to mediate both membrane insertion and excision allowed us to compare these two fundamental membrane transactions and to show that receptor-triggered insertion is a more complex process than excision from membranes.     

Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F(+) cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.     

The mechanism of bacterial infection by filamentous phages involves molecular interactions between TolA and phage protein 3 domains

The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.     

Crystal Structures of a CTXφ pIII Domain Unbound and in Complex with a Vibrio cholerae TolA Domain Reveal Novel Interaction Interfaces

Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXφ, that integrates as a prophage into the V. cholerae chromosome. CTXφ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXφ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXφ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXφ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXφ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXφ converts a harmless marine microbe into a deadly human pathogen.     

Reprogramming the infection mechanism of a filamentous phage

When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.     

Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII.

Filamentous phage assemble at the membrane of infected cells. The phage filament is released from the membrane at the end of assembly, after four to five copies of the minor proteins, pIII and pVI, have been added to the end of the virion. In the absence of pIII or pVI, phage filaments are not released, but remain associated with the cells. The C-terminal portion of pIII, termed the "C" domain, is required for the release of stable virions.With the use of pIII C-terminal fragments of increasing size, termination of assembly can be divided into various steps. An 83-residue fragment leads to the incorporation of pVI into the assembling phage, but does not release it from the membrane. A slightly longer fragment (93 residues) is sufficient to release the particle into the culture supernatant. However, these released particles are unstable in the detergent, sarkosyl, which does not disrupt wild-type phage. A fragment of >121 residues is needed for the particle to become detergent resistant. Thus, the C-domain can be divided into two subdomains: C2, sufficient for release, and C1, required for virion stability.A model for termination of phage assembly is proposed in which pIII and pVI dock to the membrane-associated filament and form a pre- termination complex. Then, a conformational change involving the C2 domain of pIII disrupts the hydrophobic interactions with the inner membrane, releasing the phage from the cells. The pIII-mediated release of phage from the membranes points to one possible mechanism for excision of membrane-anchored protein complexes from lipid bilayers.     

A proline switch controls folding and domain interactions in the gene-3-protein of the filamentous phage fd

The amino-terminal domains N1 and N2 of the gene-3-protein of phage fd form a bilobal structural and functional entity that protrudes from the phage tip. Domain N2 initiates the infection of Escherichia coli by binding to the F pilus. This binding results in the dissociation of the two domains and allows N1 to interact with the TolA receptor at the cell surface. The refolding of the N1-N2 fragment begins with the folding of domain N1, which takes a few milliseconds, followed by the folding of domain N2, which is complete within five minutes. The subsequent domain assembly is unusually slow and shows a time-constant of 6200 s at 25 degrees C. We found that the rate of this reaction is controlled by the trans to cis isomerization of the Gln212-Pro213 bond in the hinge subdomain of N2, a region that provides many interactions between N1 and N2 in the gene-3-protein. The substitution of Pro213 by Gly accelerated domain association 30-fold and revealed that the folding of the two individual domains and their assembly are indeed sequential steps in the refolding of the gene-3-protein. In the course of infection, the domains must separate to expose the binding site for TolA on domain N1. The kinetic block of domain reassembly caused by Pro213 isomerization could ensure that after the initial binding of N2 to the F pilus the open state persists until N1 and TolA are close enough for their mutual interaction. Pro213 isomerization might thus serve as a slow conformational switch in the function of the gene-3-protein.     

Filamentous phage infection: required interactions with the TolA protein.

Infection of Escherichia coli by the filamentous phage f1 is initiated by binding of the phage to the tip of the F conjugative pilus via the gene III protein. Subsequent translocation of phage DNA requires the chromosomally encoded TolQ, TolR, and TolA proteins, after the pilus presumably has withdrawn, bringing the phage to the bacterial surface. Of these three proteins, TolA is proposed to span the periplasm, since it contains a long helical domain (domain II), which connects a cytoplasmic membrane anchor domain (domain I) to the carboxyl-terminal domain (domain III). By using a transducing phage, the requirement for TolA in an F+ strain was found to be absolute. The role of TolA domains II and III in the infective process was examined by analyzing the ability of various deletion mutants of tolA to facilitate infection. The C-terminal domain III was shown to be essential, whereas the polyglycine region separating domains I and II could be deleted with no effect. Deletion of helical domain II reduced the efficiency of infection, which could be restored to normal by retaining the C-terminal half of domain II. Soluble domain III, expressed in the periplasm but not in the cytoplasm or in the medium, interfered with infection of a tolA+ strain. The essential interaction of TolA domain III with phage via gene III protein appears to require interaction with a third component, either the pilus tip or a periplasmic entity.     

Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.

BACKGROUND: Infection of male Escherichia coli cells by filamentous Ff bacteriophages (M13, fd, and f1) involves interaction of the phage minor coat gene 3 protein (g3p) with the bacterial F pilus (primary receptor), and subsequently with the integral membrane protein TolA (coreceptor). G3p consists of three domains (N1, N2, and CT). The N2 domain interacts with the F pilus, whereas the N1 domain--connected to N2 by a flexible glycine-rich linker and tightly interacting with it on the phage--forms a complex with the C-terminal domain of TolA at later stages of the infection process. RESULTS: The crystal structure of the complex between g3p N1 and TolA D3 was obtained by fusing these domains with a long flexible linker, which was not visible in the structure, indicating its very high disorder and presumably a lack of interference with the formation of the complex. The interface between both domains, corresponding to approximately 1768 A2 of buried molecular surface, is clearly defined. Despite the lack of topological similarity between TolA D3 and g3p N2, both domains interact with the same region of the g3p N1 domain. The fold of TolA D3 is not similar to any previously known protein motifs. CONCLUSIONS: The structure of the fusion protein presented here clearly shows that, during the infection process, the g3p N2 domain is displaced by the TolA D3 domain. The folds of g3p N2 and TolA D3 are entirely different, leading to distinctive interdomain contacts observed in their complexes with g3p N1. We can now also explain how the interactions between the g3p N2 domain and the F pilus enable the g3p N1 domain to form a complex with TolA.     

Structural and energetic basis of infection by the filamentous bacteriophage IKe

Filamentous phage use the two N‐terminal domains of their gene‐3‐proteins to initiate infection of Escherichia coli. One domain interacts with a pilus, and then the other domain binds to TolA at the cell surface. In phage fd, these two domains are tightly associated with each other, which renders the phage robust but non‐infectious, because the TolA binding site is inaccessible. Activation for infection requires partial unfolding, domain disassembly and prolyl isomerization. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus‐ and TolA‐binding domains of phage IKe are independent of each other in stability and folding. The site for TolA binding is thus always accessible, but the affinity is very low. The structures of the two domains, analysed by X‐ray crystallography and by NMR spectroscopy, revealed a unique fold for the N‐pilus‐binding domain and a conserved fold for the TolA‐binding domain. The absence of an activation mechanism as in phage fd and the low affinity for TolA probably explain the low infectivity of phage IKe. They also explain why, in a previous co‐evolution experiment with a mixture of phage fd and phage IKe, all hybrid phage adopted the superior infection mechanism of phage fd.     

Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins

The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.     

Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function

Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F(+) and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F(+). The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics.     

The folding mechanism of a two-domain protein: folding kinetics and domain docking of the gene-3 protein of phage fd

The gene-3 protein (G3P) of filamentous phages is essential for the infection of Escherichia coli. The carboxy-terminal domain anchors this protein in the phage coat, whereas the two amino-terminal domains N1 and N2 protrude from the phage surface. We analyzed the folding mechanism of the two-domain fragment N1-N2 of G3P (G3P(*)) and the interplay between folding and domain assembly. For this analysis, a variant of G3P(*) was used that contained four stabilizing mutations (IIHY-G3P(*)). The observed refolding kinetics extend from 10 ms to several hours. Domain N1 refolds very rapidly (with a time constant of 9.4 ms at 0.5 M guanidinium chloride, 25 degrees C) both as a part of IIHY-G3P(*) and as an isolated protein fragment. The refolding of domain N2 is slower and involves two reactions with time constants of seven seconds and 42 seconds. These folding reactions of the individual domains are followed by a very slow, spectroscopically silent docking process, which shows a time constant of 6200 seconds. This reaction was detected by a kinetic unfolding assay for native molecules. Before docking, N1 and N2 unfold fast and independently, after docking they unfold slowly in a correlated fashion. A high energy barrier is thus created by domain docking, which protects G3P kinetically against unfolding. The slow domain docking is possibly important for the infection of E.coli by the phage. Upon binding to the F pilus, the N2 domain separates from N1 and the binding site for TolA on domain N1 is exposed. Since domain reassembly is so slow, this binding site remains accessible until pilus retraction has brought N1 close to TolA on the bacterial surface.     

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.     

Energetic Communication between Functional Sites of the Gene-3-Protein during Infection by Phage fd

To initiate infection of Escherichia coli, phage fd uses its gene-3-protein (G3P) to bind first to an F pilus and then to the TolA protein at the cell surface. G3P is normally auto-inhibited because a tight interaction between the two N-terminal domains N1 and N2 buries the TolA binding site. Binding of N2 to the pilus activates G3P by initiating long-range conformational changes that are relayed to the domain interface and to a proline timer. We discovered that the 23–28 loop of the N1 domain is critical for propagating these conformational signals. The analysis of the stability and the folding dynamics of G3P variants with a shortened loop combined with TolA interaction studies and phage infection experiments reveal how the contact between the N2 domain and the 23–28 loop of N1 is energetically linked with the interdomain region and the proline timer and how it affects phage infectivity. Our results illustrate how conformational transitions and prolyl cis/trans isomerization can be coupled energetically and how conformational signals to and from prolines can be propagated over long distances in proteins.     

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality.Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells,followed by fusion of virus-cell membrane also mediated by GP.Using an human immunodeficiency virus (HIV)-based pseudotyping system,the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions.We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry.An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry.It was found that R64 and K95 are involved in receptor binding.In contrast,some residues such as I170 are important for viral entry,but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data,suggesting that these residues are involved in post-binding steps of viral entry.Furthermore,our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.     

Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability.

Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F' pilus and the periplasmic protein TolA. Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F' pilus. In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2-217) from filamentous phage fd to 1.9 A resolution and compared it to the recently published structure of the same fragment from the related Ff phage M13. While the structure of individual domains D1 and D2 of the two phages are very similar (rms<0.7 A), there is comparatively poor agreement for the overall D1D2 structure (rms>1.2 A). This is due to an apparent movement of domain D2 with respect to D1, which results in a widening of the inter-domain groove compared to the structure of the homologous M13 protein. The movement of D2 can be described as a rigid-body rotation around a hinge located at the end of a short anti-parallel beta-sheet connecting domains D1 and D2. Structural flexibility of at least parts of the D1D2 structure was also suggested by studying the thermal unfolding of g3p: the TolA binding site on D1, while fully blocked by D2 at 37 degrees C, becomes accessible after incubation at temperatures as low as 45 degrees C. Our results support a model for the early steps of phage infection whereby exposure of the coreceptor binding site on D1 is facilitated by a conformational change in the D1D2 structure, which in vivo is induced by binding to the F' pilus on the host cell and which can be mimicked in vitro by thermal unfolding.     

Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.     

A conformational unfolding reaction activates phage fd for the infection of Escherichia coli

Unfolding usually leads to the loss of the biological function of a protein. Here, we show that an unfolding reaction activates the gene-3-protein of the filamentous phage fd for its function during the infection of Escherichia coli. Before infection, the gene-3-protein is in a fully folded locked form, in which the binding site for the phage receptor TolA is buried at the domain interface. To expose this binding site, the gene-3-protein must be activated, and previously we identified the cis-to-trans isomerization at Pro213 in the hinge region between the two domains as a key step of activation. We now report that Pro213 isomerization destabilizes the protein and leads to a loss of folded structure, presumably in the hinge region. The partially unfolded form of the gene-3-protein is metastable, and trans-Pro213 arrests the protein in this activated form for an extended time, long enough to find the receptor TolA. The partial unfolding and its timing by prolyl isomerization are essential for the biological function.     

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appea...