Norbert Garnier

Insight into Signal Transduction: Structural Alterations in Transmembrane Helices Probed by Multi-1 ns Molecular Dynamics Simulations

The hypothesis of structural alteration in transmembrane helices for signal transduction process is viewed by molecular dynamics simulation techniques. For the c-erbB-2 transmembrane domain involved in oncogenicity, the occurrence of conformational changes has been previously described as transition from the alpha to pi helix. This dynamical feature is thoroughly analyzed for the wild phenotype and oncogenic sequences from a series of 18 simulations carried out on one nanosecond time scale. We show that these structural events do not depend upon the conditions of simulations like force field or starting helix coordinates. We demonstrate that the oncogenic mutations Val659 Glu, Gln and Asp do not prevent the transition. Furthermore, we show that beta branched residues, in conjunction with Gly residues in the c-erbB-2 sequence, act as destabilizers for the alpha helix structure, pi deformations are tightly related to other local structural motifs found in soluble and membrane proteins. These structural alterations are discussed in term of structure-activity relationships for the c-erbB-2 activating mechanism mediated by transmembrane domain dimerization.

TIME RESOLVED FLUORESCENCE PROPERTIES OF PHENYLALANINE IN DIFFERENT ENVIRONMENTS. COMPARISON WITH MOLECULAR DYNAMICS SIMULATION

Time resolved fluorescence of the phenylalanine residue (Phe) alone and included in the transmembrane domain (TMD) sequences of the epidermal growth factor receptor (EGFR) and ErbB-2 was studied using the synchrotron radiation source of light, and compared to molecular dynamics (MD) simulations. The fluorescence intensity decay is strongly sensitive to the environment. A mono-exponential decay was obtained for Phe amino acid alone in two different solvents and for Phe included in EGFR transmembrane sequence, with fluorescence lifetime values varying from 1.7 ns (EGFR) to 7.4 ns (Phe dissolved in water). In ErbB-2 transmembrane sequence three lifetimes were detected. The relative amplitude of the shortest one (0.14 ns) is smaller than 10%, whereas the others (0.6 and 2.2 ns) are almost equally represented. They have been attributed to different rotamers exchanging slowly. This interpretation is supported by MD simulations which evidence transitions in time series of the chi 1 dihedral angle of Phe observed in the case of ErbB-2. The anisotropy decays are similar for both peptides and indicate the presence of a correlation time in the nanosecond range (1-4 ns) and the probable existence of a very fast one (< 0.05 ns). Autocorrelation functions computed from MD simulations corroborate these results.

Transmembrane domain targeting peptide antagonizing ErbB2/Neu inhibits breast tumor growth and metastasis.

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.

Influence of a Mutation in the Transmembrane Domain of the pl85c-erbB2 Oncogene-Encoded Protein Studied by Molecular Dynamics Simulations

The c-erbB2 proto-oncogene encodes for a protein of 185kDa (p185) which becomes transforming upon the Val-->Glu transmembrane amino acid substitution. The transforming ability seems to be due to a substitution-resulting constitutive activation of the tyrosine kinase cytosolic domain of the protein. These observations prompted us to evaluate the structural and dynamical behavior of the transmembrane region of the wild and transforming p185 protein in order to understand the role of this region in the transduction mechanism. 160 ps molecular dynamics simulations in vacuo have been performed on two peptides corresponding to the sequence [651-679] of p185c-erbB2 protein and its transforming mutant Val659-->Glu659. These two sequences include the transmembrane domain and are initially postulated to be in an alpha-helix conformation. Noticeable differences in the flexibility of the two peptides are shown. The nontransforming sequence seems rather flexible and several conformational changes are detected at the junction of the mutation point [658-659] and at position Val665-Val666 during the 160 ps simulations. On the contrary, no transitions were observed for the mutated sequence which adopts a stable alpha-helix conformation. This difference in flexibility could be hypothesized as a factor involved in the regulation of the tyrosine kinase activity of p185c-erbB2.

Insight into the recognition patterns of the ErbB receptor family transmembrane domains: heterodimerization models through molecular dynamics search

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.

Molecular Dynamics Simulations of the Transmembrane Domain of the Oncogenic ErbB2 Receptor Dimer in a DMPC Bilayer

Abstract 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 α-π 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.

Transmembrane helix packing of ErbB/Neu receptor in membrane environment: a molecular dynamics study.

Abstract 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 NeuTM 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.

Ligand entry pathways in the ligand binding domain of PPARγ receptor

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.

Transmembrane peptides from tyrosine kinase receptor. Mutation-related behavior in a lipid bilayer investigated by molecular dynamics simulations.

Abstract Polar mutations in transmembrane α 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 it 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.

Inhibition of PlexA1-mediated brain tumor growth and tumor-associated angiogenesis using a transmembrane domain targeting peptide

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.