The significance of PTEN's protein phosphatase activity

Many hundreds of research papers over the last ten years have established the significance of PTEN's lipid phosphatase activity in mediating many of its effects on specific cellular processes in many different cell types, including cell growth, proliferation, survival, and migration ([Backman et al., 2002], [Iijima et al., 2002], [Leslie and Downes, 2002] and [Salmena et al., 2008]). In some cases, detailed signalling mechanisms have been identified by which these PtdInsP₃-dependent effects are manifest ([Kolsch et al., 2008], [Manning and Cantley, 2007] and [Tee and Blenis, 2005]). Further, in some settings, in vivo data from, for example genetic deletion of PTEN, relates closely with independent manipulation of the PI3K/Akt signalling pathway ([Bayascas et al., 2005], [Chen et al., 2006], [Crackower et al., 2002] and [Ma et al., 2005]). Together these studies indicate that the dominant effects of PTEN function are mediated through its regulation of PtdInsP₃-dependent signalling, but that its protein phosphatase activity also contributes in some settings. These conclusions are of great importance given the intense efforts underway to develop PI3K (EC 2.7.1.153) inhibitors as cancer therapeutics. The experiments reviewed here have firmly established that the protein phosphatase activity of PTEN plays a role in the regulation of cellular processes including migration. On the other hand, it has not been established beyond doubt that PTEN acts on substrates other than itself; no such substrates have been confidently identified and effector mechanisms for PTEN's protein phosphatase activity are currently unclear. The goal for future research must be firstly to understand the signalling mechanisms by which PTEN protein phosphatase activity acts: whether this is through identifying substrates, or working out how autodephosphorylation mediates its effects. Secondly, and critically, the significance of PTEN's protein phosphatase activity must be established in vivo. This can be achieved through relating the phenotypes intervening with both PTEN and with protein phosphatase effector pathways when they are identified, and through the generation of mouse models expressing substrate selective PTEN mutants. We should then be able to answer the important question of whether PTEN's protein phosphatase activity contributes to tumour suppression.     

Crystal Structure of a Chimeric Receptor Binding Protein Constructed from Two Lactococcal Phages

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry to manufacture cheeses, is subject to infection by a diverse population of virulent phages. We have previously determined the structures of three receptor binding proteins (RBPs) from lactococcal phages TP901-1, p2, and bIL170, each of them having a distinct host range. Virulent phages p2 and bIL170 are classified within the 936 group, while the temperate phage TP901-1 is a member of the genetically distinct P335 polythetic group. These RBPs comprise three domains: the N-terminal domain, binding to the virion particle; a β-helical linker domain; and the C-terminal domain, bearing the receptor binding site used for host recognition. Here, we have designed, expressed, and determined the structure of an RBP chimera in which the N-terminal and linker RBP domains of phage TP901-1 (P335) are fused to the C-terminal RBP domain of phage p2 (936). This chimera exhibits a stable structure that closely resembles the parental structures, while a slight displacement of the linker made RBP domain adaptation efficient. The receptor binding site is structurally indistinguishable from that of native p2 RBP and binds glycerol with excellent affinity.A broad number of products are manufactured by large-scale bacterial fermentation, including the value-added fermented dairy products. Most bacterial fermentation industries have experienced problems with phage contamination. Phage outbreaks are costly and time-consuming because they can slow or arrest the fermentation process and adversely affect product quality (15). For decades, the dairy industry has relied on an array of strategies to control this natural phenomenon, including rotation of their bacterial cultures (11, 24, 25). However, in spite of these efforts, new virulent lactococcal phages keep emerging. A better understanding of the various mechanisms affecting the genetic diversity of the phage population is necessary for optimal phage control strategies (18).Lactococcal phages are among the most studied bacterial viruses because of the economic importance of their hosts. Hundreds of lactococcal phages have been isolated, and the vast majority of them have a long, contractile tail, thereby belonging to the Siphoviridae family (1). Lactococcus lactis phages are currently classified into 10 genetically distinct groups (10), but only members of 3 of them are highly adapted to multiply in milk, namely, the 936, c2, and P335 groups (11, 24, 25). The first step for such an effective viral infection is host recognition, which necessitates the interaction between the adsorption device located at the distal tail end of the phage and the cell surface receptor (32). Members of the 936 and P335 groups recognize their host through an interaction between their receptor binding protein (RBP) (13) and receptors, probably lipoteichoic acids, at the host cell surface (27, 29-31).We have previously determined the crystal structures of three RBPs, from the virulent lactococcal phages p2 (30, 31) and bIL170 (936 group) (27) and from the temperate phage TP901-1 (P335 group) (29). The RBPs of these phages have a similar architecture of three protomers related by a threefold axis. Each protomer comprises three domains: the N terminus (named shoulders in p2), the interlaced β-prism linker (the “neck” domain), and the jelly-roll domain (2) at the C terminus (the “head” domain). This last domain harbors a saccharide binding site likely involved in host recognition, as it binds with high affinity to phosphoglycerol, a component of teichoic acid (8, 19, 27, 29-31). We have previously shown that the shoulder and neck domains are highly conserved in the RBPs of 936-like phages (8, 19, 27, 29-31). The individuality of the RBP C-terminal domain sequence likely dictates phage specificity for the receptor, which may specifically recognize different substitutions (H, GlcNAc, or d-Ala) of the phosphoglycerol moieties of the L. lactis teichoic acid polymers. Recently, the complete genomic sequence of the reference virulent phage P335 was determined, and comparative analysis revealed that the C terminus of its RBP showed homology to the RBP of the virulent lactococcal phage P475 of the 936 group (17). Such homology between RBP head domains was surprising because the two lactococcal phage groups rarely shared common genes or domains. This observation suggested that modular shuffling of domains can occur between these otherwise genetically distinct phage groups.The overall fold of the N-terminal RBP domain is different in 936- and P335-like phages. In the P335 group, the N-terminal domain comprises a unique helix that fits into the rest of the phage baseplate (28, 29) (Fig. ​(Fig.1A),1A), while in the 936 group, this 140-residue domain is a large β-sandwich with an external α-helix (30) (Fig. ​(Fig.1B).1B). Nonetheless, the N-terminal domains of the two RBPs may still be, related because both appear to be built using a coiled coil, although the 936-like phages have an additional β-sandwich. The β-prism linkers (neck domain) of the two phage groups also differ in sequence and in radius, but they have a similar fold, the latter being also close to that of T4 phage short fiber (33). The linker domain of phage TP901-1 is wider than that of p2 and exhibits a repeated motif (G-X-Y-X-Y, where X is polar and Y nonpolar). Finally, the C-terminal domains of both species share the same fold, a jelly-roll motif (2) also found in adenovirus (5) and reovirus (3, 4, 6).Open in a separate windowFIG. 1.Structures and sequences of RBPs from lactococcal phages. (A) Three-dimensional structure of the RBP from phage TP901-1 (P335 group; blue). (B) Three-dimensional structure of the RBP from phage p2 (936 group; magenta). (C) View of a model associating domains of TP901-1 (N terminus and linker domain, below red line, blue) and p2 (head, above red line, magenta) RBPs. (D) Three-dimensional crystal structure of chimera form 1 (yellow) assembled according to the model in panel C. (E) Sequence alignment of the RBPs of p2 (part) and TP901-1. The secondary structure is described above the alignment. The binding residues are shown with blue dots. The hinge proline (Pro 162/63) is identified by a red arrow. The chimera is composed of the N-terminal domain (residues 17 to 33) and the linker domain residues (residues 34 to 63) from phage TP901-1 RBP and the C-terminal domain (residues 163 to 264) from phage p2 RBP.The question addressed here was whether exchange between the C-terminal domains of two phage groups would lead to a stable protein with conserved binding capacity. To answer this question, we have generated an RBP chimera comprising the N-terminal and linker domains of phage TP901-1 fused to the C-terminal domain of phage p2. We have produced this chimera and determined its crystal structure and its sugar binding capacity. These results indicate that straightforward domain exchange produced a stable chimera with a conserved binding capacity and a structure close to that of each of the parental parts.     

Crystal Structure and Function of a DARPin Neutralizing Inhibitor of Lactococcal Phage TP901-1: COMPARISON OF DARPin AND CAMELID VHH BINDING MODE*

Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppU·BppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 Å resolution. The DARPin·RBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry for the production of an array of fermented milk products. Several industrial strains are sensitive to various distinct bacteriophages, mostly belonging to the Siphoviridae family. The lactococcal phage population is divided in at least 10 genetically distinct groups, of which the 936, c2, and P335 groups are prominent (1, 2). These L. lactis-infecting phages are considerably problematic in causing milk fermentation failures and resulting in decreased yields as well as low quality products (3). Preventing these infections has proven to be difficult because of lactococcal phage ubiquity, biodiversity, and genomic plasticity (4).Phage infection is initiated by binding of the phage receptor-binding protein (RBP),⁵ located within the baseplate at the distal part of the tail, to its receptor on the host cell surface (5). We have previously solved the crystal structures of the three RBPs of the lactococcal phages p2 (936) (6), bIL170 (936) (7), TP901-1 (P335) (8), and their chimera (9) as well as characterized their saccharide binding sites (10). The RBPs of these phages have a similar homotrimeric architecture related by a 3-fold axis. They comprise three domains: the N terminus shoulder domain, the interlaced β-prism neck domain, and the jellyroll head domain at the C terminus. The head domain has a saccharide binding site likely involved in host recognition. The lactococcal phage TP901-1 contains a double-disk-shaped baseplate at the tip of its tail which is made of a lower baseplate protein (BppL) and an upper baseplate protein (BppU) (11).One strategy to minimize bacteriophage infections is to competitively block phage adsorption by adding a protein that specifically binds to the phage RBP. A neutralizing llama VHH domain recognizing the head domain of the phage p2 RBP has been used to block L. lactis phage infection in milk fermentation (12). Lactococcal phages could readily escape neutralization by generating mutations interfering with VHH binding over the large interaction surface while keeping the central polysaccharide receptor binding pocket intact (10). Designed ankyrin repeat proteins (DARPins) may be another tool to neutralize viral infection, as they display distinct characteristics from VHHs and contain the required properties in terms of stability and facility of expression (13).Ankyrin repeat proteins are found in virtually all phyla and mediate specific protein-protein interactions in all cell compartments (14). The ankyrin elementary module is composed of 33 amino acids structured as a β-turn followed by two antiparallel α-helices and a loop connected to the β-turn of the next repeat. The repeats are stacked in a rigid manner. In creating a DARPin library, residues in each repeat were subdivided in two groups; (i) randomized residues constituting potential target interaction points and (ii) framework residues, important for maintaining the ankyrin fold (13). Libraries with varying repeat numbers were assembled and named according to the constituent repeat number; N2C and N3C libraries were used in this study, with two and three internal repeats inserted between the N and C capping repeats, respectively. DARPins are a powerful alternative to the use of antibodies, notably because of their very high expression rates in Escherichia coli, their high stability paired with high affinity, and successful reports of their use in co-crystallization (15–19). Their architecture results in a very rigid structure that facilitates multiple crystal contacts and may promote crystal formation of the protein of interest by providing additional surfaces for such crystal contacts.We report here the selection and analysis of DARPin binders directed against a macromolecular multiprotein ensemble, the TP901-1 baseplate BppU·BppL protein complex. Ribosome display selection, ELISA screening, and surface plasmon resonance (SPR) measurements allowed us to isolate and characterize three N2C DARPins that recognized the RBP (BppL of the BppU·BppL complex) with high specificity and affinity. Further studies showed that the three DARPins bound to a unique area of the RBP at the tip of the head domain. QELS, MALS, UV, and refractometry coupled online with a size exclusion chromatography (SEC) column allowed us to monitor complex formation in solution as well as to estimate DARPin binding stoichiometry. Crystals of one of these selected DARPins in complex with the RBP were obtained, and the x-ray structure was solved at 2.1 Å resolution. This constitutes the first structure of a DARPin complex originating from the N2C library and the highest resolution for a DARPin complex structure reported to date. Finally, phage adsorption inhibition experiments demonstrated that the three N2C DARPins strongly inhibited L. lactis infection by TP901-1. We describe the DARPin·RBP interface and compare it to other DARPin interfaces. We also compare it to the p2 RBP·VHH5 complex, a previously selected llama VHH domain inhibiting p2 phage adsorption (12), to highlight the different binding mode of these two types of binders.