ABSTRACT
The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.
Subject(s)
BTB-POZ Domain , Transcription Factors , Xenopus Proteins , Animals , Humans , Cell Nucleus/metabolism , Cell Nucleus/genetics , Crystallography, X-Ray , HEK293 Cells , Models, Molecular , Protein Binding , Protein Multimerization , Repressor Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/chemistry , Transcription Factors/metabolism , Transcription Factors/genetics , Xenopus laevis , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus Proteins/chemistryABSTRACT
Despite significant progress in cancer imaging and treatment over the years, early diagnosis and metastasis detection remain a challenge. Molecular magnetic resonance imaging (MRI), with its high resolution, can be well adapted to fulfill this need, requiring the design of contrast agents which target specific tumor biomarkers. Netrin-1 is an extracellular protein overexpressed in metastatic breast cancer and implicated in tumor progression and the appearance of metastasis. This study focuses on the design and preclinical evaluation of a novel Netrin-1-specific peptide-based MRI probe, GdDOTA-KKTHDAVR (Gd-K), to visualize metastatic breast cancer. The targeting peptide sequence was identified based on the X-ray structure of the complex between Netrin-1 and its transmembrane receptor DCC. Molecular docking simulations support the probe design. In vitro studies evidenced submicromolar affinity of Gd-K for Netrin-1 (KD = 0.29 µM) and good MRI efficacy (proton relaxivity, r1 = 4.75 mM-1 s-1 at 9.4 T, 37 °C). In vivo MRI studies in a murine model of triple-negative metastatic breast cancer revealed successful tumor visualization at earlier stages of tumor development (smaller tumor volume). Excellent signal enhancement, 120% at 2 min and 70% up to 35 min post injection, was achieved (0.2 mmol/kg injected dose), representing a reasonable imaging time window and a superior contrast enhancement in the tumor as compared to Dotarem injection.
Subject(s)
Biomarkers, Tumor , Triple Negative Breast Neoplasms , Mice , Humans , Animals , Molecular Probes , Netrin-1 , Molecular Docking Simulation , Contrast Media/chemistry , Peptides , Magnetic Resonance Imaging/methodsABSTRACT
Protein SUMOylation is a ubiquitylation-like post-translational modification (PTM) that is synthesized through an enzymatic cascade involving an E1 (SAE1:SAE2), an E2 (UBC9), and various E3 enzymes. In the final step of this process, the small ubiquitin-like modifier (SUMO) is transferred from the UBC9â¼SUMO thioester onto a lysine residue of a protein substrate. This reaction can be accelerated by an E3 ligase. As the UBC9â¼SUMO thioester is chemically unstable, a stable mimetic is desirable for structural studies of UBC9â¼SUMO alone and in complex with a substrate and/or an E3 ligase. Recently, a strategy for generating a mimetic of the yeast E2â¼SUMO thioester by mutating alanine 129 of Ubc9 to a lysine has been reported. Here, we reproduce and further investigate this approach using the human SUMOylation system and characterize the resulting mimetic of human UBC9â¼SUMO1. We show that substituting lysine for alanine 129, but not for other active-site UBC9 residues, results in a UBC9 variant that is efficiently auto-SUMOylated. The auto-modification is dependent on cysteine 93 of UBC9, suggesting that it proceeds via this residue, through the same pathway as that for SUMOylation of substrates. The process is also partially dependent on aspartate 127 of UBC9 and accelerated by high pH, highlighting the importance of the substrate lysine protonation state for efficient SUMOylation. Finally, we present the crystal structure of the UBC9-SUMO1 molecule, which reveals the mimetic in an open conformation and its polymerization via the noncovalent SUMO-binding site on UBC9. Similar interactions could regulate UBC9â¼SUMO in some cellular contexts.
Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin , Humans , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin/metabolism , Lysine/metabolism , Ubiquitin-Protein Ligases/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , SumoylationABSTRACT
DNA glycosylases are emerging as relevant pharmacological targets in inflammation, cancer and neurodegenerative diseases. Consequently, the search for inhibitors of these enzymes has become a very active research field. As a continuation of previous work that showed that 2-thioxanthine (2TX) is an irreversible inhibitor of zinc finger (ZnF)-containing Fpg/Nei DNA glycosylases, we designed and synthesized a mini-library of 2TX-derivatives (TXn) and evaluated their ability to inhibit Fpg/Nei enzymes. Among forty compounds, four TXn were better inhibitors than 2TX for Fpg. Unexpectedly, but very interestingly, two dithiolated derivatives more selectively and efficiently inhibit the zincless finger (ZnLF)-containing enzymes (human and mimivirus Neil1 DNA glycosylases hNeil1 and MvNei1, respectively). By combining chemistry, biochemistry, mass spectrometry, blind and flexible docking and X-ray structure analysis, we localized new TXn binding sites on Fpg/Nei enzymes. This endeavor allowed us to decipher at the atomic level the mode of action for the best TXn inhibitors on the ZnF-containing enzymes. We discovered an original inhibition mechanism for the ZnLF-containing Fpg/Nei DNA glycosylases by disulfide cyclic trimeric forms of dithiopurines. This work paves the way for the design and synthesis of a new structural class of inhibitors for selective pharmacological targeting of hNeil1 in cancer and neurodegenerative diseases.
Subject(s)
DNA Glycosylases/antagonists & inhibitors , DNA Glycosylases/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Purines/chemistry , Purines/pharmacology , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/pharmacology , Bacteria/enzymology , Binding Sites , Crystallography, X-Ray , DNA Repair , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , Structure-Activity Relationship , Thioxanthenes/chemistry , Thioxanthenes/pharmacologyABSTRACT
In the early stage of a drug discovery process, the selection and optimization of a ligand is mainly based on equilibrium thermodynamic constants such as KD or IC50 values, which are representatives of the affinity of the compound for its target. However, these criteria are not able to correctly evaluate the efficacy of compounds in vivo and result in many failures of compound development during phase II of clinical trials. Residence time (RT) is an important parameter associated to an in vivo drug's safety and efficacy. The determination or modulation of kinetic rates correlated to RT may be performed to identify the best drug candidates in the early stages of a drug design project. For this purpose, a number of experimental methodologies were developed but remain costly in both time and money. Herein, we developed a novel protocol based on biased molecular dynamics simulations and transition-state theory in order to predict relative ligand kinetic rates and relative RTs of a series of compounds. First, we have repeatedly simulated the unbinding process of the ligand from its binding site to the outside of the target. Next, we sample the conformational space along the determined unbinding paths to allow relevant statistical distributions of the system. The free energy profiles associated to these distributions are then computed and used to predict the kinetics parameters. The studied set was composed of eight ligands with fast, intermediate, and slow dissociation rates and binding to the active and inactive states of p38α protein kinase. The proposed method provides an excellent correlation between the predicted values and the experimentally measured kinetic rates, in addition to a detailed characterization of the kinetic paths at the atomic level.
Subject(s)
Protein Kinase Inhibitors/chemistry , Ligands , Molecular Dynamics Simulation , Protein Binding , Protein Kinase Inhibitors/pharmacology , ThermodynamicsABSTRACT
The neuropilin-plexin receptor complex regulates tumor cell migration and proliferation and thus is an interesting therapeutic target. High expression of neuropilin-1 is indeed associated with a bad prognosis in glioma patients. Q-RTPCR and tissue-array analyses showed here that Plexin-A1 is highly expressed in glioblastoma and that the highest level of expression correlates with the worse survival of patients. We next identified a developmental and tumor-associated pro-angiogenic role of Plexin-A1. Hence, by using molecular simulations and a two-hybrid like assay in parallel with biochemical and cellular assays we developed a specific Plexin-A1 peptidic antagonist disrupting transmembrane domain-mediated oligomerization of the receptor and subsequent signaling and functional activity. We found that this peptide exhibits anti-tumor activity in vivo on different human glioblastoma models including glioma cancer stem cells. Thus, screening Plexin-A1 expression and targeting Plexin-A1 in glioblastoma patients exhibit diagnostic and therapeutic value.
Subject(s)
Antineoplastic Agents/pharmacology , Brain Neoplasms/pathology , Glioma/pathology , Neovascularization, Pathologic/prevention & control , Nerve Tissue Proteins/antagonists & inhibitors , Peptides/pharmacology , Receptors, Cell Surface/antagonists & inhibitors , Animals , Biomarkers, Tumor/metabolism , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Chick Embryo , Chorioallantoic Membrane/metabolism , Glioblastoma/metabolism , Glioblastoma/pathology , Glioma/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Nerve Tissue Proteins/metabolism , Protein Domains , Receptors, Cell Surface/metabolism , Tissue Array Analysis , ZebrafishABSTRACT
Breast cancer is still a deadly disease despite major achievements in targeted therapies designed to block ligands or ligand-binding subunits of major tyrosine kinase receptors. Relapse is significant and metastases deleterious, which demands novel strategies for fighting this disease. Here, we report a proof-of-concept experiment demonstrating that small peptides interfering with the transmembrane domain of the tyrosine kinase epidermal growth factor receptor ErbB2 exhibit anticancer properties when used at micromolar dosages in a genetically engineered mouse model of breast cancer. Different assays demonstrate the specificity of the ErbB2-targeting peptide, which induces long-term reduction of ErbB2 phosphorylation and Akt signaling consistent with reduced tumor cell proliferation and increased survival. Microcomputed tomography analysis established the antimetastatic activity of the peptide and its impact on primary tumor growth. This reveals the interior of the cell membrane as an unexplored dimension for drug design.
Subject(s)
Receptor, ErbB-2/metabolism , Amino Acid Sequence , Animals , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Kaplan-Meier Estimate , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/pathology , Lung Neoplasms/secondary , MCF-7 Cells , Mice , Mice, Transgenic , Molecular Sequence Data , Peptides/chemistry , Peptides/therapeutic use , Peptides/toxicity , Phosphorylation/drug effects , Protein Multimerization , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-2/genetics , Tomography, X-Ray ComputedABSTRACT
The x-ray structure of NccX, a type II transmembrane metal sensor, from Cupriavidus metallidurans 31A has been determined at a resolution of 3.12 Å. This was achieved after solubilization by dodecylphosphocholine and purification in the presence of the detergent. NccX crystal structure did not match the model based on the extensively characterized periplasmic domain of its closest homologue CnrX. Instead, the periplasmic domains of NccX appeared collapsed against the hydrophobic transmembrane segments, leading to an aberrant topology incompatible with membrane insertion. This was explained by a detergent-induced redistribution of the hydrophobic interactions among the transmembrane helices and a pair of hydrophobic patches keeping the periplasmic domains together in the native dimer. Molecular dynamics simulations performed with the full-length protein or with the transmembrane segments were used along with in vivo homodimerization assays (TOXCAT) to evaluate the determinants of the interactions between NccX protomers. Taken as a whole, computational and experimental results are in agreement with the structural model of CnrX where a cradle-shaped periplasmic metal sensor domain is anchored into the inner membrane by two N-terminal helices. In addition, they show that the main determinant of NccX dimerization is the periplasmic soluble domain and that the interaction between transmembrane segments is highly dynamic. The present work introduces a new crystal structure for a transmembrane protein and, in line with previous studies, substantiates the use of complementary theoretical and in vivo investigations to rationalize a three-dimensional structure obtained in non-native conditions.
Subject(s)
Bacterial Proteins/chemistry , Cupriavidus/metabolism , Detergents/chemistry , Membrane Proteins/chemistry , Metalloproteins/chemistry , Amino Acid Sequence , Crystallization , Crystallography, X-Ray , Gene Expression Regulation, Bacterial , Molecular Dynamics Simulation , Molecular Sequence Data , Mutation , Protein Multimerization , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
The cancer associated class 3 semaphorins require direct binding to neuropilins and association to plexins to trigger cell signaling. Here, we address the role of the transmembrane domains of neuropilin 1 and plexin A1 for the dimerization of the two receptors by characterizing the assembly in lipid bilayers using coarse-grained molecular dynamics simulations. From experimental evidence using a two-hybrid system showing the biochemical association of the two receptors transmembrane domains, we performed molecular simulations in DOPC and POPC demonstrating spontaneously assembly to form homodimers and heterodimers with a very high propensity for right-handed packing of the helices. Inversely, left-handed packing was observed with a very low propensity. This mode of packing was observed uniquely when the plexin A1 transmembrane domain was involved in association. Potential of mean force calculations were used to predict a hierarchy of self-association for the monomers: the two neuropilin 1 transmembrane domains strongly associated, neuropilin 1 and plexin A1 transmembrane domains associated less and the two plexin A1 transmembrane domains weakly but significantly associated. We demonstrated that homodimerization and heterodimerization are driven by GxxxG motifs, and that the sequence context modulates the packing mode of the plexin A1 transmembrane domains. This work presents major advances towards our understanding of membrane signaling platforms assembly through membrane domains and provides exquisite information for the design of antagonist drugs defining a novel class of therapeutic agents.
Subject(s)
Cell Membrane/metabolism , Molecular Dynamics Simulation , Nerve Tissue Proteins/metabolism , Neuropilin-1/metabolism , Receptors, Cell Surface/metabolism , Semaphorins/metabolism , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Nerve Tissue Proteins/chemistry , Neuropilin-1/chemistry , Phosphatidylcholines/metabolism , Protein Multimerization , Protein Structure, Quaternary , Receptors, Cell Surface/chemistry , ThermodynamicsABSTRACT
Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31-53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.
Subject(s)
Blood Proteins/chemistry , Blood Proteins/metabolism , Galectins/chemistry , Galectins/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cell Survival , Cells, Cultured , Cytotoxins/chemistry , Cytotoxins/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mitochondria/metabolism , Models, Molecular , Molecular Sequence Data , Sequence AlignmentABSTRACT
To address the question of ligand entry process, we report targeted molecular dynamics simulations of the entry of the flexible ionic ligand GW0072 in the ligand binding domain of the nuclear receptor PPARγ. Starting with the ligand outside the receptor the simulations led to a ligand docked inside the binding pocket resulting in a structure very close to the holo-form of the complex. The results showed that entry process is guided by hydrophobic interactions and that entry pathways are very similar to exit pathways. We suggest that TMD method may help in discriminating between ligands generated by in silico docking.
Subject(s)
Molecular Dynamics Simulation , PPAR gamma/chemistry , PPAR gamma/metabolism , Ligands , Protein Binding , Protein Structure, Tertiary , ThermodynamicsABSTRACT
The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα2, EcHUß2, and EcHUαß) are in thermal equilibrium between two dimeric conformations (N2 â I2) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N2 to I2 thermal transition of the EcHUß2 homodimer. On the basis of these data, a realistic 3D model is proposed for the major I2 conformation of EcHUß2. This model is in agreement with previous experimental data.
Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , Escherichia coli Proteins/chemistry , Molecular Dynamics Simulation , Protein Denaturation , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Hot Temperature , Magnetic Resonance Spectroscopy , Protein Conformation , Time FactorsABSTRACT
The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that gamma-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604-612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.
Subject(s)
DNA, Bacterial/radiation effects , DNA-Binding Proteins/radiation effects , Escherichia coli Proteins/radiation effects , Gamma Rays , Lac Repressors/radiation effects , Molecular Dynamics Simulation , Amino Acid Sequence , Base Sequence , Binding Sites/physiology , Binding Sites/radiation effects , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Dihydroxyphenylalanine/chemistry , Dihydroxyphenylalanine/metabolism , Dihydroxyphenylalanine/radiation effects , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Hydrogen Bonding , Lac Repressors/chemistry , Lac Repressors/metabolism , Models, Molecular , Molecular Sequence Data , Operator Regions, Genetic , Oxidation-Reduction , Static ElectricityABSTRACT
ErbB receptors undergo a complex interaction network defining hierarchical and competition relationships. Dimerization is driven entirely by receptor-receptor interactions and the transmembrane domains play a role in modulating the specificity and the selection of the partners during signal transduction. To shed light on the role of the GxxxG-like dimerization motifs in the formation of ErbB transmembrane heterodimers, we propose structural models resulting from conformational search method combined with molecular dynamics simulations. Left-handed structures of the transmembrane heterodimers are found preponderant over right-handed structures. All heterotypic heterodimers undergo two modes of association either via the N-terminal motif or the C-terminal motif. The transmembrane domain of ErbB3 impairs this C-terminal motif but also associates with the other partners owing to the presence of Gly residues. The two dimerization modes involve different orientations of the two helices. Thus, a molecular-switch model allowing the transition between the two dimerizing states may apply to the heterodimers and could help interpret receptor competition for the formation of homodimers and heterodimers. The comparison between experimental and theoretical results on the dimerization hierarchy of the transmembrane domains is not straightforward. However, we demonstrate that the intrinsic properties of the transmembrane sequences are an important component in heterodimer formation and that the ErbB2 and ErbB3 transmembrane domains have a strong power for heterodimerization as observed experimentally.
Subject(s)
Membrane Microdomains/chemistry , Models, Chemical , Models, Molecular , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/ultrastructure , Receptor, ErbB-3/chemistry , Receptor, ErbB-3/ultrastructure , Binding Sites , Computer Simulation , Dimerization , Membrane Microdomains/ultrastructure , Protein Binding , Protein Conformation , Protein Structure, TertiaryABSTRACT
Computational methods are useful to identify favorable structures of transmembrane (TM) helix oligomers when experimental data are not available or when they cannot help to interpret helix-helix association. We report here a global search method using molecular dynamics (MD) simulations to predict the structures of transmembrane homo and heterodimers. The present approach is based only on sequence information without any experimental data and is first applied to glycophorin A to validate the protocol and to the HER2-HER3 heterodimer receptor. The method successfully reproduces the experimental structures of the TM domain of glycophorin A (GpA(TM)) with a root mean square deviation of 1.5 A. The search protocol identifies three energetically stable models of the TM domain of HER2-HER3 receptor with favorable helix-helix arrangement, including right-handed and left-handed coiled-coils. The predicted TM structures exhibit the GxxxG-like motif at the dimer interface which is presumed to drive receptor oligomerization. We demonstrate that native structures of TM domain can be predicted without quantitative experimental data. This search protocol could help to predict structures of the TM domain of HER heterodimer family.
Subject(s)
Glycophorins/chemistry , Protein Conformation , Amino Acid Sequence , Computational Biology , Energy Transfer , Membranes/chemistry , Models, Molecular , Molecular Sequence Data , Oncogene Proteins v-erbB/chemistryABSTRACT
Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains. Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the Neu(TM) helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization-competent structure responsible of the proper topology necessary for receptor activation.
Subject(s)
Receptor, ErbB-2/chemistry , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Computer Simulation , Dimerization , Dimyristoylphosphatidylcholine/chemistry , Lipid Bilayers , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Models, Molecular , Phosphatidylcholines/chemistry , Protein Conformation , Receptor, ErbB-2/metabolismABSTRACT
Polar mutations in transmembrane alpha helices may alter the structural details of the hydrophobic sequences and control intermolecular contacts. We have performed molecular dynamics simulations on the transmembrane domain of the proto-oncogenic and the oncogenic forms of the Neu receptor in a fluid DMPC bilayer to test whether the Glu mutation which replaces the Val residue at position 664 may alter the helical structure and its insertion in the membrane. The simulations show that the wild and the mutant forms of the transmembrane domain have a different behavior in the bilayer. The native transmembrane sequence is found to be more flexible than in the presence of the Glu mutation, characterized by a tendency to pi deformation to accommodate the helix length to the membrane thickness. The mutant form of this domain does not evidence helical deformation in the present simulation. Hydrophobic matching is achieved both by a larger helix tilt and a vertical shift of the helix towards the membrane interface, favoring the accessibility of the Glu side chain to the membrane environment. A rapid exchange of hydrogen bond interactions with the surrounding water molecules and the lipid headgroups is observed. The difference in the behavior between the two peptides in a membrane environment was also observed experimentally. Both simulation and experimental results agree with the hypothesis that water may act as an intermediate for the formation of cross links between the facing Glu side chains stabilizing the dimer.
Subject(s)
Lipid Bilayers/metabolism , Mutation , Peptides/chemistry , Receptor Protein-Tyrosine Kinases/chemistry , Amino Acid Sequence , Dimerization , Dimyristoylphosphatidylcholine/chemistry , Glutamic Acid/chemistry , Hydrogen Bonding , Indicators and Reagents/chemistry , Lipid Bilayers/chemistry , Lipids/chemistry , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Receptor, ErbB-2/chemistry , Software , Time Factors , Valine/chemistryABSTRACT
The critical Val/Glu mutation in the membrane spanning domain of the rat Neu receptor confers the ability for ligand-independent signaling and leads to increased dimerization and transforming ability. There is evidence that the two transmembrane interacting helices play a role in receptor activation by imposing orientation constraints to the intracellular tyrosine kinase domains. By using MD simulations we have attempted to discriminate between correct and improper helix-helix packing by examining the structural and energetic properties of preformed left-handed and right-handed structures in a fully hydrated DMPC bilayer. The best energetic balance between the residues at the helix-helix interface and the residues exposed to the lipids is obtained for helices in symmetrical left-handed interactions packed together via Glu side chain/Ala backbone interhelical hydrogen bonds. Analyses demonstrate the importance of the ATVEG motif in helix-helix packing and point to additional contacting residues necessary for association. Our findings, all consistent with experimental data, suggest that a symmetrical left-handed structure of the helices could be the transmembrane domain configuration that promotes receptor activation and transformation. The present study may provide further insight into signal transduction mechanisms of the ErbB/Neu receptors.
Subject(s)
Dimyristoylphosphatidylcholine/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/metabolism , Computer Simulation , Dimerization , Models, Molecular , Protein Structure, TertiaryABSTRACT
Molecular dynamics simulations of an atomic model of the transmembrane domain of the oncogenic ErbB2 receptor dimer embedded in an explicit dimyristoylphosphatidylcholine (DMPC) bilayer were performed for more than 4 ns. The oncogenic Glu mutation in the membrane spanning segment plays a major role in tyrosine kinase activity and receptor dimerization, and is thought to be partly responsible for the structure of the transmembrane domain of the active receptor. MD results show that the interactions between the two transmembrane helices are characteristic of a left-handed packing as previously demonstrated from in vacuo simulations. Moreover, MD results reveal the absence of persistent hydrogen bonds between the Glu side chains in a membrane environment, which raise the question of the ability for Glu alone to stabilize the TM domain of the ErbB2 receptor. Interestingly the formation of the alpha-pi motif in the two ErbB2 transmembrane helices confirms the concept of intrinsic sequence-induced conformational flexibility. From a careful analysis of our MD results, we suggest that the left-handed helix-helix packing could be the key to correctly orient the intracellular domain of the activated receptor dimer. The prediction of such interactions from computer simulations represents a new step towards the understanding of signaling mechanisms.