Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

In vitro and in vivo anti-tumor effect of Trichobakin fused with urokinase-type plasminogen activator ATF-TBK.

BACKGROUND: Trichobakin (TBK), a member of type I ribosome-inactivating proteins (RIPs), was first successfully cloned from Trichosanthes sp Bac Kan 8-98 in Vietnam. Previous study has shown that TBK acts as a potential protein synthesis inhibitor; however, the inhibition efficiency and specificity of TBK on cancer cells remain to be fully elucidated. METHODS AND RESULTS: In this work, we employed TBK and TBK conjugated with a part of the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), which contains the Ω-loop that primarily interacts with urokinase-type plasminogen activator receptor, and can be a powerful carrier in the drug delivery to cancer cells. Four different human tumor cell lines and BALB/c mice bearing Lewis lung carcinoma cells (LLC) were used to evaluate the role of TBK and ATF-TBK in the inhibition of tumor growth. Here we showed that the obtained ligand fused RIP (ATF-TBK) reduced the growth of four human cancer cell lines in vitro in the uPA receptor level-dependent manner, including the breast adenocarcinoma MDA-MB 231 cells and MCF7 cells, the prostate carcinoma LNCaP cells and the hepatocellular carcinoma HepG2 cells. Furthermore, the conjugate showed anti-tumor activity and prolonged the survival time of tumor-bearing mice. The ATF-TBK also did not cause the death of mice with doses up to 48 mg/kg, and they were not significantly distinct on parameters of hematology and serum biochemistry between the control and experiment groups. CONCLUSIONS: In conclusion, ATF-TBK reduced the growth of four different human tumor cell lines and inhibited lung tumor growth in a mouse model with little side effects. Hence, the ATF-TBK may be a target to consider as an anti-cancer agent for clinical trials.

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.

Atomistic mechanism of the constitutive activation of PDGFRA via its transmembrane domain.

Single-point mutations in the transmembrane (TM) region of receptor tyrosine kinases (RTKs) can lead to abnormal ligand-independent activation. We use a combination of computational modeling, NMR spectroscopy and cell experiments to analyze in detail the mechanism of how TM domains contribute to the activation of wild-type (WT) PDGFRA and its oncogenic V536E mutant. Using a computational framework, we scan all positions in PDGFRA TM helix for identification of potential functional mutations for the WT and the mutant and reveal the relationship between the receptor activity and TM dimerization via different interfaces. This strategy also allows us design a novel activating mutation in the WT (I537D) and a compensatory mutation in the V536E background eliminating its constitutive activity (S541G). We show both computationally and experimentally that single-point mutations in the TM region reshape the TM dimer ensemble and delineate the structural and dynamic determinants of spontaneous activation of PDGFRA via its TM domain. Our atomistic picture of the coupling between TM dimerization and PDGFRA activation corroborates the data obtained for other RTKs and provides a foundation for developing novel modulators of the pathological activity of PDGFRA.

The Conformation of the Epidermal Growth Factor Receptor Transmembrane Domain Dimer Dynamically Adapts to the Local Membrane Environment.

The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kinases, mediating a variety of cellular responses in normal biological processes and in pathological states of multicellular organisms. Different modes of dimerization of the human EGFR transmembrane domain (TMD) in different membrane mimetics recently prompted us to propose a novel signal transduction mechanism based on protein-lipid interaction. However, the experimental evidence for it was originally obtained with slightly different TMD fragments used in the two different mimetics, compromising the validity of the comparison. To eliminate ambiguity, we determined the nuclear magnetic resonance (NMR) structure of the bicelle-incorporated dimer of the EGFR TMD fragment identical to the one previously used in micelles. The NMR results augmented by molecular dynamics simulations confirm the mutual influence of the TMD and lipid environment, as is required for the proposed lipid-mediated activation mechanism. They also reveal the possible functional relevance of a subtle interplay between the concurrent processes in the lipid and protein during signal transduction.

HER2 Transmembrane Domain (TMD) Mutations (V659/G660) That Stabilize Homo- and Heterodimerization Are Rare Oncogenic Drivers in Lung Adenocarcinoma That Respond to Afatinib.

INTRODUCTION: Erb-b2 receptor tyrosine kinase (HER2) transmembrane domain (TMD) mutations (HER2V659E, HER2G660D) have previously been identified in lung adenocarcinomas, but their frequency and clinical significance is unknown. METHODS: We prospectively analyzed 8551 consecutive lung adenocarcinomas using hybrid capture-based comprehensive genomic profiling (CGP) at the request of the individual treating physicians for the purpose of making therapy decisions. RESULTS: We identified 15 cases (0.18%) of HER2 TMD mutations (HER2V659E/D, HER2G660D) through CGP of 8551 lung adenocarcinomas. HER2 TMD mutations were mutually exclusive from HER2 kinase domain mutations and other oncogenic drivers in lung adenocarcinoma. Only two cases with HER2 TMD mutations (13%) had concurrent Erb-b2 receptor tyrosine kinase 2 gene (HER2) amplification. Structural analysis of HER2 TMD association revealed that mutations at positions V659 and G660 to the highly polar residues glutamic acid, aspartic acid, or arginine should stabilize homodimerization and heterodimerization of HER2 in the active conformation. Treatment with afatinib, a pan-HER inhibitor, resulted in durable clinical response in three of four patients with lung adenocarcinoma, with two harboring HER2V659E and one with double HER2V659E/G660R mutations. HER2 TMD mutations (V659 and G660) are found in other non-NSCLC malignancies, and analogous TMD mutations are also found in EGFR, HER3, and HER4. CONCLUSION: HER2 TMD mutations represent rare but distinct targetable driver mutations in lung adenocarcinoma. CGP capable of detecting diverse HER2 alterations, including HER2 TMD mutations, should be broadly adopted to identify all patients who may benefit from HER2-targeted therapies.

Alternative packing of EGFR transmembrane domain suggests that protein-lipid interactions underlie signal conduction across membrane.

The human epidermal growth factor receptor (EGFR) of HER/ErbB receptor tyrosine kinase family mediates a broad spectrum of cellular responses transducing biochemical signals via lateral dimerization in plasma membrane, while inactive receptors can exist in both monomeric and dimeric forms. Recently, the dimeric conformation of the helical single-span transmembrane domains of HER/ErbB employing the relatively polar N-terminal motifs in a fashion permitting proper kinase activation was experimentally determined. Here we describe the EGFR transmembrane domain dimerization via an alternative weakly polar C-terminal motif A(661)xxxG(665) presumably corresponding to the inactive receptor state. During association, the EGFR transmembrane helices undergo a structural adjustment with adaptation of inter-molecular polar and hydrophobic interactions depending upon the surrounding membrane properties that directly affect the transmembrane helix packing. This might imply that signal transduction through membrane and allosteric regulation are inclusively mediated by coupled protein-protein and protein-lipid interactions, elucidating paradoxically loose linkage between ligand binding and kinase activation.

HER2 Transmembrane Domain Dimerization Coupled with Self-Association of Membrane-Embedded Cytoplasmic Juxtamembrane Regions.

Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family transduce biochemical signals across plasma membrane, playing a significant role in vital cellular processes and in various cancers. Inactive HER/ErbB receptors exist in equilibrium between the monomeric and unspecified pre-dimerized states. After ligand binding, the receptors are involved in strong lateral dimerization with proper assembly of their extracellular ligand-binding, single-span transmembrane, and cytoplasmic kinase domains. The dimeric conformation of the HER2 transmembrane domain that is believed to support the cytoplasmic kinase domain configuration corresponding to the receptor active state was previously described in lipid bicelles. Here we used high-resolution NMR spectroscopy in another membrane-mimicking micellar environment and identified an alternative HER2 transmembrane domain dimerization coupled with self-association of membrane-embedded cytoplasmic juxtamembrane region. Such a dimerization mode appears to be capable of effectively inhibiting the receptor kinase activity. This finding refines the molecular mechanism regarding the signal propagation steps from the extracellular to cytoplasmic domains of HER/ErbB receptors.

ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom.

Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 µg/g in experiments on house fly Musca domestica larvae and with LD50 20 µg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended ß-hairpin loop and short ß-hairpin loop which are capable of making "scissors-like mutual motions".

The Membrane Mimetic Affects the Spatial Structure and Mobility of EGFR Transmembrane and Juxtamembrane Domains.

The epidermal growth factor receptor (EGFR) is one of the most extensively studied receptor tyrosine kinases, as it is involved in a wide range of cellular processes and severe diseases. Recent works reveal that the single-helix transmembrane domains and cytoplasmic juxtamembrane regions play an important role in the receptor activation process. Here we present the results of our investigation of the spatial structure and mobility of the EGFR transmembrane domain and juxtamembrane regions in various membranelike environments, which shed light on the effects of the membrane physical properties and composition on the behavior of the juxtamembrane domain.

Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies.

Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.

Role of dimerization efficiency of transmembrane domains in activation of fibroblast growth factor receptor 3.

Mutations in transmembrane (TM) domains of receptor tyrosine kinases are shown to cause a number of inherited diseases and cancer development. Here, we use a combined molecular modeling approach to understand molecular mechanism of effect of G380R and A391E mutations on dimerization of TM domains of human fibroblast growth factor receptor 3 (FGFR3). According to results of Monte Carlo conformational search in the implicit membrane and further molecular dynamics simulations, TM dimer of this receptor is able to form a number of various conformations, which differ significantly by the free energy of association in a full-atom model bilayer. The aforementioned mutations affect dimerization efficiency of TM segments and lead to repopulation of conformational ensemble for the dimer. Particularly, both mutations do not change the dimerization free energy of the predominant (putative "non-active") symmetric conformation of TM dimer, while affect dimerization efficiency of its asymmetric ("intermediate") and alternative symmetric (putative "active") models. Results of our simulations provide novel atomistic prospective of the role of G380 and A391E mutations in dimerization of TM domains of FGFR3 and their consecutive contributions to the activation pathway of the receptor.

Structural and thermodynamic insight into the process of "weak" dimerization of the ErbB4 transmembrane domain by solution NMR.

Specific helix-helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure-function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651-678)(2) form a right-handed parallel dimer through the N-terminal double GG4-like motif A(655)GxxGG(660) in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer-dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true ("ideal") solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix-helix interactions, which may be critical for membrane protein activity.

Spatial structure of the transmembrane domain heterodimer of ErbB1 and ErbB2 receptor tyrosine kinases.

Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed alpha-helical bundle through their N-terminal double GG4-like motif T(648)G(649)X(2)G(652)A(653) and glycine zipper motif T(652)X(3)S(656)X(3)G(660), respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.

Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state.

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.

Antiamoebin I in methanol solution: rapid exchange between right-handed and left-handed 3(10)-helical conformations.

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe(1)-Aib-Aib-Aib-Iva-Gly-Leu-Aib(8)-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl(16). By using the uniformly (13)C,(15)N-labeled sample of Aam-I, the set of conformationally dependent J couplings and (3h)J(NC) couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the (13)C(beta) nuclei (Deltadelta((13)C(beta))) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe(1)-Gly(6)) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3(10)-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib(8) residue. The (15)N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib(8) residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic 'propensity' possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.

Unique dimeric structure of BNip3 transmembrane domain suggests membrane permeabilization as a cell death trigger.

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.

New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1.

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.