Interaction of huntingtin fragments with brain membranes--clues to early dysfunction in Huntington's disease

Abstract Huntingtin is a large, multi-domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N-terminus causes Huntington's disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo- and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N-terminal huntingtin fragment accumulated in postsynaptic membranes during the pre-symptomatic period of 4-8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA- and AMPA-receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N-terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild-type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline-rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis.     

Huntingtin disrupts lipid bilayers in a polyQ-length dependent manner

Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 N-terminal amino acids of htt have been shown to further modulate aggregation. Additionally, these 17 amino acids appear to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-lengths (35Q, 46Q, 51Q, and myc-53Q) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. Furthermore, the addition of an N-terminal myc-tag to the htt exon1 fragments impeded the interaction of htt with the bilayer.     

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in β cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.     

Autoinhibition of Endophilin in Solution via Interdomain Interactions

Endophilin is a key protein involved in clathrin-mediated endocytosis. Previous computational and experimental work suggested that the N-terminal helix is embedded into the membrane to induce curvature; however, the role of the SH3 domain remains controversial. To address this issue, we performed computer simulations of the endophilin dimer in solution to understand the interaction between the N-BAR and SH3 domains and its effect on biological function. We predict that the helix binds to the SH3 domain through hydrophobic and salt-bridge interactions. This protects the hydrophobic residues on both domains and keeps the SH3 domain near the end of the N-BAR domain, in agreement with previous experimental results. The complex has a binding strength similar to a few hydrogen bonds (13.0 ± 0.6 kcal/mol), and the SH3 domain stabilizes the structure of the N-terminal helix in solution. Electrostatic calculations show a large region of strongly positive electrostatic potential near the N-terminal that can orient the helix toward the membrane and likely embed the helix into the membrane surface. This predicted mechanism suggests that endophilin can select for both curvature and electrostatic potential when interacting with membranes, highlighting the importance of the SH3 domain in regulating the function of endophilin.     

Characterization of Endophilin B1b,a brain-specific membrane-associated lysophosphatidic acid acyl transferase with properties distinct from endophilin A1

We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.     

Phosphoinositide 3-kinase regulates plasma membrane targeting of the Ras-specific exchange factor RasGRP1

Receptor-induced targeting of exchange factors to specific cellular membranes is the predominant mechanism for initiating and compartmentalizing signal transduction by Ras GTPases. The exchange factor RasGRP1 has a C1 domain that binds the lipid diacylglycerol and thus can potentially mediate membrane localization in response to receptors that are coupled to diacylglycerol-generating phospholipase Cs. However, the C1 domain is insufficient for targeting RasGRP1 to the plasma membrane. We found that a basic/hydrophobic cluster of amino acids within the plasma membrane-targeting domain of RasGRP1 is instead responsible for plasma membrane targeting. This basic/hydrophobic cluster binds directly to phospholipid vesicles containing phosphoinositides via electrostatic interactions with polyanionic phosphoinositide headgroups and insertion of a tryptophan into the lipid bilayer. B cell antigen receptor ligation and other stimuli induce plasma membrane targeting of RasGRP1 by activating the phosphoinositide 3-kinase signaling pathway, which generates phosphoinositides within the plasma membrane. Direct detection of phosphoinositides by the basic/hydrophobic cluster of RasGRP1 provides a novel mechanism for coupling and co-compartmentalizing phosphoinositide 3-kinase and Ras signaling and, in coordination with diacylglycerol detection by the C1 domain, gives RasGRP1 the potential to serve as an integrator of converging signals from the phosphoinositide 3-kinase and phospholipase C pathways.     

Molecular modeling of the membrane targeting of phospholipase C pleckstrin homology domains

Phospholipases C (PLCs) reversibly associate with membranes to hydrolyze phosphatidylinositol-4, 5-bisphosphate (PI[4,5]P(2)) and comprise four main classes: beta, gamma, delta, and epsilon. Most eukaryotic PLCs contain a single, N-terminal pleckstrin homology (PH) domain, which is thought to play an important role in membrane targeting. The structure of a single PLC PH domain, that from PLCdelta1, has been determined; this PH domain binds PI(4,5)P(2) with high affinity and stereospecificity and has served as a paradigm for PH domain functionality. However, experimental studies demonstrate that PH domains from different PLC classes exhibit diverse modes of membrane interaction, reflecting the dissimilarity in their amino acid sequences. To elucidate the structural basis for their differential membrane-binding specificities, we modeled the three-dimensional structures of all mammalian PLC PH domains by using bioinformatic tools and calculated their biophysical properties by using continuum electrostatic approaches. Our computational analysis accounts for a large body of experimental data, provides predictions for those PH domains with unknown functions, and indicates functional roles for regions other than the canonical lipid-binding site identified in the PLCdelta1-PH structure. In particular, our calculations predict that (1). members from each of the four PLC classes exhibit strikingly different electrostatic profiles than those ordinarily observed for PH domains in general, (2). nonspecific electrostatic interactions contribute to the membrane localization of PLCdelta-, PLCgamma-, and PLCbeta-PH domains, and (3). phosphorylation regulates the interaction of PLCbeta-PH with its effectors through electrostatic repulsion. Our molecular models for PH domains from all of the PLC classes clearly demonstrate how a common structural fold can serve as a scaffold for a wide range of surface features and biophysical properties that support distinctive functional roles.     

Ponticulin, a developmentally-regulated plasma membrane glycoprotein, mediates actin binding and nucleation

Ponticulin is a 17,000-dalton transmembrane glycoprotein that is involved in the binding and nucleation of actin filaments by Dictyostelium discoideum plasma membranes. The major actin-binding protein isolated from these membranes by F-actin affinity chromatography, ponticulin also binds F-actin on blot overlays. The actin-binding activity of ponticulin in vitro is identical to that observed for purified plasma membranes: it resists extraction with 0.1 N NaOH, is sensitive to high salt concentrations, and is destroyed by heat, proteolysis, and thiol reduction and alkylation. A cytoplasmic domain of ponticulin mediates binding to actin because univalent antibody fragments directed against the cytoplasmic surface of this protein inhibit 96% of the actin-membrane binding in sedimentation assays. Antibody specific for ponticulin removes both ponticulin and the ability to reconstitute actin nucleation activity from detergent extracts of solubilized plasma membranes. Levels of plasma membrane ponticulin increase 2- to 3-fold during aggregation streaming, when cells adhere to each other and are highly motile. Although present throughout the plasma membrane, ponticulin is preferentially localized to some actin-rich membrane structures, including sites of cell-cell adhesion and arched regions of the plasma membrane reminiscent of the early stages of pseudopod formation. Ponticulin also is present but not obviously enriched at phagocytic cups of log-phase amebae. These results indicate that ponticulin may function in vivo to attach and nucleate actin filaments at the cytoplasmic surface of the plasma membrane. A 17,000-dalton analogue of ponticulin has been identified in human polymorphonuclear leukocyte plasma membranes by immunoblotting and immunofluorescence microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyglutamine expansion in huntingtin alters its interaction with phospholipids

Huntingtin has an expanded polyglutamine tract in patients with Huntington's disease. Huntingtin localizes to intracellular and plasma membranes but the function of huntingtin at membranes is unknown. Previously we reported that exogenously expressed huntingtin bound pure phospholipids using protein-lipid overlays. Here we show that endogenous huntingtin from normal ( Hdh ^7Q/7Q) mouse brain and mutant huntingtin from Huntington's disease ( Hdh ^140Q/140Q) mouse brain bound to large unilamellar vesicles containing phosphoinositol (PI) PI 3,4-bisphosphate, PI 3,5-bisphosphate, and PI 3,4,5-triphosphate [PI(3,4,5)P3]. Huntingtin interactions with multivalent phospholipids were similar to those of dynamin. Mutant huntingtin associated more with phosphatidylethanolamine and PI(3,4,5)P3 than did wild-type huntingtin, and associated with other phospholipids not recognized by wild-type huntingtin. Wild-type and mutant huntingtin also bound to large unilamellar vesicles containing cardiolipin, a phospholipid specific to mitochondrial membranes. Maximal huntingtin-phospholipid association required inclusion of huntingtin amino acids 171–287. Endogenous huntingtin recruited to the plasma membrane in cells that incorporated exogenous PI 3,4-bisphosphate and PI(3,4,5)P3 or were stimulated by platelet-derived growth factor or insulin growth factor 1, which both activate PI 3-kinase. These data suggest that huntingtin interacts with membranes through specific phospholipid associations and that mutant huntingtin may disrupt membrane trafficking and signaling at membranes.     

Investigation into the mechanism regulating MRP localization

The major PKC substrates MARCKS and MacMARCKS (MRP) are membrane-binding proteins implicated in cell spreading, integrin activation and exocytosis. According to the myristoyl-electrostatic switch model the co-operation between the myristoyl moiety and the positively charged effector domain (ED) is an essential mechanism by which proteins bind to membranes. Loss of the electrostatic interaction between the ED and phospholipids, such as Ptdins(4,5)P2, results in the translocation of such proteins to the cytoplasm. While this model has been extensively tested for the binding of MARCKS far less is known about the mechanisms regulating MRP localization. We demonstrate that after phosphorylation, MRP is relocated to the intracellular membranes of late endosomes and lysosomes. MRP binds to all membranes via its myristoyl moiety, but for its localization at the plasma membrane the ED is also required. Although the ED of MRP can bind to Ptdins(4,5)P2 in vitro, this binding is not essential for its retention at or targeting to the plasma membrane. We conclude that the co-operation between the myristoyl moiety and the ED is not required for the binding to membranes in general but that it is essential for the targeting of MRP to the plasma membrane in a Ptdins(4,5)P2-independent manner.     

Implicit solvent simulations of peptide interactions with anionic lipid membranes

A recently developed implicit membrane model (IMM1) is supplemented with a Gouy-Chapman term describing counterion-screened electrostatic interactions of a solute with negatively charged membrane lipids. The new model is tested on peptides that bind to anionic membranes. Pentalysine binds just outside the plane of negative charge, whereas Lys-Phe peptides insert their aromatic rings into the hydrophobic core. Melittin and magainin 2 bind more strongly to anionic than to neutral membranes and in both cases insert their hydrophobic residues into the hydrocarbon core. The third domain of Antennapedia homeodomain (penetratin) binds as an alpha-helix in the headgroup region. Cardiotoxin II binds strongly to anionic membranes but marginally to neutral ones. In all cases, the location and configuration of the peptides are consistent with experimental data, and the effective energy changes upon binding compare favorably with experimental binding free energies. The model opens the way to exploring the effect of membrane charge on the location, conformation, and dynamics of a large variety of biologically active peptides on membranes.     

Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.     

N-terminal PDZ domain is required for NHERF dimerization

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.     

The intrinsically disordered nuclear localization signal and phosphorylation segments distinguish the membrane affinity of two cytidylyltransferase isoforms

Membrane phosphatidylcholine homeostasis is maintained in part by a sensing device in the key regulatory enzyme, CTP:phosphocholine cytidylyltransferase (CCT). CCT responds to decreases in membrane phosphatidylcholine content by reversible membrane binding and activation. Two prominent isoforms, CCTα and -β2, have nearly identical catalytic domains and very similar membrane binding amphipathic helical (M) domains but have divergent and structurally disordered N-terminal (N) and C-terminal phosphorylation (P) regions. We found that the binding affinity of purified CCTβ2 for anionic membranes was weaker than CCTα by more than an order of magnitude. Using chimeric CCTs, insertion/deletion mutants, and truncated CCTs, we show that the stronger affinity of CCTα can be attributed in large part to the electrostatic membrane binding function of the polybasic nuclear localization signal (NLS) motif, present in the unstructured N-terminal segment of CCTα but lacking in CCTβ2. The membrane partitioning of CCTβ2 in cells enriched with the lipid activator, oleic acid, was also weaker than that of CCTα and was elevated by incorporation of the NLS motif. Thus, the polybasic NLS can function as a secondary membrane binding motif not only in vitro but in the context of cell membranes. A comparison of phosphorylated, dephosphorylated, and region P-truncated forms showed that the in vitro membrane affinity of CCTβ2 is more sensitive than CCTα to phosphorylation status, which antagonizes membrane binding of both isoforms. These data provide a model wherein the primary membrane binding motif, an amphipathic helical domain, works in collaboration with other intrinsically disordered segments that modulate membrane binding strength. The NLS reinforces, whereas the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.     

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50–60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm² in the search for a second endosome with which to interact.     

Phosphatidylinositol 3-phosphate induces the membrane penetration of the FYVE domains of Vps27p and Hrs

The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain.     

N-Terminal Proteolysis of Full-Length Mutant Huntingtin in an Inducible PC12 Cell Model of Huntington’s Disease

Proteolytic cleavage of mutant huntingtin may play a key role in the pathogenesis of Huntington’s disease; however the steps in huntingtin proteolysis are not fully understood. Huntingtin was shown to be cleaved by caspases and calpains within a region between 460-600 amino acids from the N-terminus. Two smaller N-terminal fragments produced by unknown protease have been previously described as cp-A and cp-B. To further investigate the huntingtin proteolytic pathway, we used an inducible PC12 cell model expressing full-length huntingtin with either normal or expanded polyglutamine. This cell model recapitulates several steps of huntingtin proteolysis: proteolysis mediated by caspases within the region previously mapped for caspase cleavage, and cleavage generating two novel N-terminal fragments (cp-1 approximately 90-105 residues long and cp-2 extending beyond 115-129 epitope of huntingtin). Interestingly, the deletion of amino acids 105-114 (mapped previously as a cleavage site for cp-A) failed to affect the production of cp-1 or cp-2. Therefore, we conclude that these new fragments are distinct from previously described cp-A and cp-B. We demonstrate that cp-1 and cp-2 fragments are produced and accumulate within nuclear and cytoplasmic inclusions prior to huntingtin-induced cell toxicity, and these fragments can be formed by caspase-independent proteolytic cleavage of huntingtin in PC12 cells. In addition, inhibition of calpains leads to decreased subsequent degradation of cp-1 and cp-2 fragments, and accelerated formation of inclusions. Further delineation of huntingtin cleavage events may lead to novel therapeutic targets for HD.     

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor

Two human rhinovirus serotypes complexed with two- and five-domain soluble fragments of the cellular receptor, intercellular adhesion molecule-1, have been investigated by X-ray crystallographic analyses of the individual components and by cryo-electron microscopy of the complexes. The three-dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N-terminal domain of the receptor binds to the rhinovirus 'canyon' surrounding the icosahedral 5-fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N-terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.     

Behavior of the N-terminal helices of the diphtheria toxin T domain during the successive steps of membrane interaction

During intoxication of a cell, the translocation (T) domain of the diphtheria toxin helps the passage of the catalytic domain across the membrane of the endosome into the cytoplasm. We have investigated the behavior of the N-terminal region of the T domain during the successive steps of its interaction with membranes at acidic pH using tryptophan fluorescence, its quenching by brominated lipids, and trypsin digestion. The change in the environment of this region was monitored using mutant W281F carrying a single native tryptophan at position 206 at the tip of helix TH1. The intrinsic propensity to interact with the membrane of each helix of the N-terminus of the T domain, TH1, TH2, TH3, and TH4, was also studied using synthetic peptides. We showed the N-terminal region of the T domain was not involved in the binding of the domain to the membrane, which occurred at pH 6 mainly through hydrophobic effects. At that stage of the interaction, the N-terminal region remained strongly solvated. Further acidification eliminated repulsive electrostatic interactions between this region and the membrane, allowing its penetration into the membrane by attractive electrostatic interactions and hydrophobic effects. The peptide study indicated the nature of forces contributing to membrane penetration. Overall, the data suggested that the acidic pH found in the endosome not only triggers the formation of the molten globule state of the T domain required for membrane interaction but also governs a progressive penetration of the N-terminal part of the T domain in the membrane. We propose that these physicochemical properties are necessary for the translocation of the catalytic domain.