Fluorescence Resonance Energy Transfer Analysis of Merlin Conformational Changes

Neurofibromatosis type 2 is an inherited autosomal disorder caused by biallelic inactivation of the NF2 tumor suppressor gene. The NF2 gene encodes a 70-kDa protein, merlin, which is a member of the ezrin-radixin-moesin (ERM) family. ERM proteins are believed to be regulated by a transition between a closed conformation, formed by binding of their N-terminal FERM domain and C-terminal tail domain (CTD), and an open conformation, in which the two domains do not interact. Previous work suggests that the tumor suppressor function of merlin is similarly regulated and that only the closed form is active. Therefore, understanding the mechanisms that control its conformation is crucial. We have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer, both as purified protein and in live cells. Using these tools, we find that merlin exists predominately as a monomer in a stable, closed conformation that is mediated by the central α-helical domain. The contribution from the FERM-CTD interaction to the closed conformation appears to be less important. Upon phosphorylation or interaction with an effector protein, merlin undergoes a subtle conformational change, suggesting a novel mechanism that modulates the interaction between the FERM domain and the CTD.Neurofibromatosis type 2 is an inherited autosomal disorder that is characterized by bilateral schwannomas of the eighth cranial nerve. The tumor suppressor gene responsible for this disorder, NF2, was cloned in 1993 (45). Biallelic inactivation of the NF2 gene is also seen in spontaneous schwannoma, meningioma, and malignant mesothelioma (22). In mouse models, deletion of the Nf2 gene is embryonic lethal, indicating an essential role for NF2 in development (24). Heterozygous mice develop a variety of aggressive metastatic tumors that have lost the wild-type allele (23). Targeted deletion of the Nf2 gene in Schwann cells leads to schwannoma formation (7). In vitro, Nf2-null cells grow to significantly higher densities (31), suggesting that contact inhibition of growth is impaired in these cells and that mediation of growth arrest at high cell density may be the basis for the tumor suppressor function of the NF2 gene. In normal fibroblasts, merlin is inactive as a growth suppressor in subconfluent cells, becoming activated as they approach confluence, thereby effecting contact inhibition of growth (26).The NF2 gene encodes a 70-kDa protein called merlin (for moesin, ezrin, radixin-like protein), which shares significant homology with members of the ezrin-radixin-moesin (ERM) branch of the Band 4.1 superfamily (45). The domain structure of merlin, also shared with other ERM proteins, consists of an N-terminal FERM domain, followed by a central α-helical region (CH) and a C-terminal tail domain (CTD). The merlin FERM domain has relatively high sequence similarity with other ERM family members, a 60 to 70% identity over the first 300 amino acids. The CH domain and the CTD show much lower identity (28 to 36%); however, the α-helical character of the CH domain is preserved, as is the heptad repeat pattern typical of α-helices that form coiled coils (46).The critical point of regulation of all the ERM proteins is a high-affinity intramolecular interaction between the C-terminal domain and the FERM domain (4) (Fig. ​(Fig.1).1). The FERM domain folds into a three-lobed cloverleaf structure that acts as a multifaceted docking site for protein binding partners (16, 39). The CTD, consisting of four major and two minor helices and a beta sheet, binds to the FERM domain by extending across the face of the F2 and F3 lobes (32). This intramolecular head-to-tail binding results in a “closed” conformation, with the C-terminal domain covering much of the surface of the FERM domain (32, 44). For ezrin, radixin and moesin, the CTD functions as a mask, blocking access of effector molecules, such as the cell surface receptors CD44 and ICAM2 and adaptor molecules, like EBP50/NHERF, to sites on the surface of the FERM domain (11, 25, 44). The interaction between the CTD and FERM domain is regulated by phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) binding to the FERM domain and by phosphorylation of a critical residue in the CTD (3, 6, 10, 49). This residue, threonine 567 in ezrin, is conserved throughout the ERM family (21). Phosphorylation introduces a negative charge and a bulky side group that effectively reduces the affinity of the interaction, releasing the CTD from the FERM domain and causing a transition to an open conformation. Low-angle rotary shadowing electron microscopy (13) and biochemical studies (12) of purified radixin suggest that in the open conformation it is an extended filamentous structure with globular N and C termini that is greater than 240 Å in length. Signal transduction systems, such as the epidermal growth factor and Rho A pathways, induce phosphorylation of ERM proteins at the conserved C-terminal threonine via a number of kinases, including Rho kinase and protein kinase Cα (21, 28). Thus, conformational regulation of ERM proteins can be a point of integration of ERM activity with signal transduction pathways. The overall concept of ERM regulation, then, is centered upon a transition between an inactive, closed conformation that is mediated by the FERM-CTD interaction and an active, open conformation that is regulated by phosphorylation. In these two states, ERM proteins likely interact with different sets of binding partners, resulting in distinct functional outcomes.Open in a separate windowFIG. 1.ERM tertiary structure as represented by the crystal structure of full-length Sf-moesin (20), but with the merlin amino acid sequence substituted for Sf-moesin. Approximate boundary amino acid residues for all domains appear at the top of the figure. Each domain is assigned a different color. The ERM structure consists of an N-terminal FERM domain folded into three lobes, F1, F2, and F3. This is followed by a central α-helical domain containing three subhelices (αA, αB, and αC) and a CTD with four short helices. An ERM protein is thought to have an open conformation, an extended structure with the FERM domain and the CTD separated by the α-helical domain, that is more than 240 Å long. In the closed conformation, the α-helical domain bends at the αA-αB junction and again at the αB-αC junction, causing the CTD to be positioned over F2 and F3 of the FERM domain. More than half of the surface of the FERM domain is masked by interaction with the CTD, αA, and parts of αB and αC.Like the classical ERMs, merlin is also thought to be regulated by changes in conformation. The FERM domain and the CTD of merlin interact with each other, albeit at a lower level of affinity than the ezrin FERM domain and the CTD (29). There are important differences, however, between merlin and the other ERM proteins. First, phosphorylation of the conserved C-tail threonine T576 has not been reported to occur in mammalian merlin, and nonphosphorylatable and phosphomimetic substitutions at this site have no effect on merlin activity (42). Instead, merlin is phosphorylated at serine 518 in the CTD, a target of the p21-activated kinase PAK and protein kinase A (1, 18, 47). The growth-suppressive function of merlin is activated by dephosphorylation of S518 by the phosphatase PP1δ in a density-dependent manner (14). Second, it was reported in a study using FERM domain and CTD truncates of merlin that only cotransfection of both the N-and C-terminal halves resulted in growth suppression (38). Together, these observations suggested a model of inactive, phosphorylated merlin in an open conformation that, upon cell-to-cell contact, is dephosphorylated and transitions to a closed, growth suppressive conformation.The existing model for conformational regulation described above is inferred from indirect data and assays that generally measure the interaction of isolated FERM and CTD truncates rather than full-length molecules (9, 29, 38). It has been impossible to test directly because tools have not been available to specifically assay for either the open or the closed form of merlin. Therefore, we have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer (FRET), both as purified protein and in live cells. Using these tools, we show that merlin exists predominately as a monomer in a stable, largely closed conformation. Additionally, we find that the closed conformation is largely mediated by the central α-helical domain; the contribution of the FERM-CTD interaction appears to be less significant than previously thought. Finally, we find that phosphorylation and protein interaction cause unexpectedly small changes in merlin conformation. We propose a new and more refined model for merlin regulation, in which merlin function is regulated by specific but subtle conformational changes that modulate the interaction between the FERM domain and the CTD.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

FERM domain phosphoinositide binding targets merlin to the membrane and is essential for its growth-suppressive function

The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP(2), via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin.     

Identification of integrin beta subunit mutations that alter affinity for extracellular matrix ligand

We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.     

Design, synthesis, biochemical studies, cellular characterization, and structure-based computational studies of small molecules targeting the urokinase receptor

The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.     

Amino acid changes in Drosophila alphaPS2betaPS integrins that affect ligand affinity

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2betaPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavbeta3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2betaPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the betaPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the betaPS I domain (betaPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2betaPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin beta subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the betaPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because betaPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.     

Modulation of ligand binding by alternative splicing of the alphaPS2 integrin subunit

The Drosophila alphaPS2 integrin subunit is found in two isoforms. alphaPS2C contains 25 residues not found in alphaPS2m8, encoded by the alternative eighth exon. Previously, it was shown that cells expressing alphaPS2C spread more effectively than alphaPS2m8 cells on fragments of the ECM protein Tiggrin, and that alphaPS2C-containing integrins are relatively insensitive to depletion of Ca(2+). Using a ligand mimetic probe for Tiggrin affinity (TWOW-1), we show that the affinity of alphaPS2CbetaPS for this ligand is much higher than that of alphaPS2m8betaPS. However, the two isoforms become more similar in the presence of activating levels of Mn(2+). Modeling indicates that the exon 8-encoded residues replace the third beta strand of the third blade of the alpha subunit beta-propeller structure, and generate an exaggerated loop between this and the fourth strand. alphaPS2 subunits with the extra loop structure but with an m8-like third strand, or subunits with a C-like strand but an m8-like short loop, both fail to show alphaPS2C-like affinity for TWOW-1. Surprisingly, a single C > m8-like change at the third strand-loop transition point is sufficient to make alphaPS2C require Ca(2+) for function, despite the absence of any known cation binding site in this region. These data indicate that alternative splicing in integrin alpha subunit extracellular domains may affect ligand affinity via relatively subtle alterations in integrin conformation. These results may have relevance for vertebrate alpha6 and alpha7, which are alternatively spliced at the same site.     

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.     

Antifouling properties of oligo(lactose)-based self-assembled monolayers

The antifouling (AF) properties of oligo(lactose)-based self-assembled monolayers (SAMs), using four different proteins, zoospores of the green alga Ulva linza and cells of the diatom Navicula incerta, were investigated. The SAM-forming alkylthiols, which contained 1, 2 or 3 lactose units, showed significant variation in AF properties, with no differences in wettability. Non-specific adsorption of albumin and pepsin was low on all surfaces. Adsorption of lysozyme and fibrinogen decreased with increasing number of lactose units in the SAM, in agreement with the generally observed phenomenon that thicker hydrated layers provide higher barriers to protein adsorption. Settlement of spores of U. linza followed an opposite trend, being greater on the bulkier, more hydrated SAMs. These SAMs are more ordered for the larger saccharide units, and it is therefore hypothesized that the degree of order, and differences in crystallinity or stiffness between the surfaces, is an important parameter regulating spore settlement on these surfaces.