The membrane topography of the diphtheria toxin T domain linked to the a chain reveals a transient transmembrane hairpin and potential translocation mechanisms.
ABSTRACT: The diphtheria toxin T domain helps translocate the A chain of the toxin across membranes. To gain insight into translocation, the membrane topography of key residues in T domain attached to the A chain (AT protein) was compared to that in the isolated T domain using fluorescence techniques. This study demonstrates that residues in T domain hydrophobic helices (TH5-TH9) tended to be less exposed to aqueous solution in the AT protein than in the isolated T domain. Under conditions in which the loop connecting TH5 to TH6/7 is located stably on the cis (insertion) side of the membrane in the isolated T domain, it moves between the cis and trans sides of the membrane in the AT protein. This is indicative of the formation of a dynamic, transient transmembrane hairpin topography by TH5-TH7 in the AT protein. Since TH8 and TH9 also form a transmembrane hairpin, this means that TH5-TH9 may form a cluster of transmembrane helices. These helices have a nonpolar surface likely to face the lipid bilayer in a helix cluster and a surface rich in uncharged hydrophilic residues which in a helix cluster would likely be facing inward (and perhaps be pore-lining). This uncharged hydrophilic surface could play a crucial role in translocation, interacting transiently with the translocating A chain. A similar motif can be found in, and may be important for, other protein translocation systems.
Project description:Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 ? helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1-TH4, translocates across the membrane to the trans side. Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane ?-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6-TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6-TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.
Project description:DesK is a Histidine Kinase that allows <i>Bacillus subtilis</i> to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.
Project description:The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism, and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3-5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately -8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between -9.5 and -12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain.
Project description:pH-induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side-chain protonation accompanying the formation of the membrane-competent state of the toxin's translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry [e.g., long-microsecond-range, all-atom molecular dynamics (MD) simulations; continuum electrostatics calculations; and thermodynamic integration (TI)] with several experimental techniques of fluorescence spectroscopy. TI calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mol), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over 8 ?s demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2 and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.
Project description:To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine approximately 6:4 POPC:cholesterol<POPC approximately dioleoylphosphatidylcholine (DOPC)<1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG)</=1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20-30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.
Project description:Autotransporter proteins are defined by the ability to drive their own secretion across the bacterial outer membrane. The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adhesion to the respiratory epithelium. In this report, we present the crystal structure of the C-terminal end of Hia, corresponding to the entire Hia translocator domain and part of the passenger domain (residues 992-1098). This domain forms a beta-barrel with 12 transmembrane beta-strands, including four strands from each subunit. The beta-barrel has a central channel of 1.8 nm in diameter that is traversed by three N-terminal alpha-helices, one from each subunit. Mutagenesis studies demonstrate that the transmembrane portion of the three alpha-helices and the loop region between the alpha-helices and the neighboring beta-strands are essential for stability of the trimeric structure of the translocator domain, and that trimerization of the translocator domain is a prerequisite for translocator activity. Overall, this study provides important insights into the mechanism of translocation in trimeric autotransporters.
Project description:The structure of a 36-amino-acid-long amino-terminal fragment of phospholamban (phospholamban[1-36]) in aqueous solution containing 30% trifluoroethanol was determined by nuclear magnetic resonance. The peptide, which comprises the cytoplasmic domain and six residues of the transmembrane domain of phospholamban, assumes a conformation characterized by two alpha-helices connected by a turn. The residues of the turn are Ile18, Glu19, Met20, and Pro21, which are adjacent to the two phosphorylation sites Ser16 and Thr17. The proline is in a trans conformation. The helix comprising amino acids 22-36 is well determined (the root mean square deviation for the backbone atoms, calculated for a family of 18 nuclear magnetic resonance structures is 0.57 A). Recently, two molecular models of the transmembrane domain of phospholamban were proposed in which a symmetric homopentamer is composed of a left-handed coiled coil of alpha-helices. The two models differ by the relative orientation of the helices. The model proposed by,Simmerman et al. (H.K. Simmerman, Y.M. Kobayashi, J.M. Autry, and L.R. Jones, 1996, J. Biol. Chem. 271:5941-5946), in which the coiled coil is stabilized by a leucine-isoleucine zipper, is similar to the transmembrane pentamer structure of the cartilage oligomeric membrane protein determined recently by x-ray (V. Malashkevich, R. Kammerer, V Efimov, T. Schulthess, and J. Engel, 1996, Science 274:761-765). In the model proposed by Adams et al. (P.D. Adams, I.T. Arkin, D.M. Engelman, and A.T. Brunger, 1995, Nature Struct. Biol. 2:154-162), the helices in the coiled coil have a different relative orientation, i.e., are rotated clockwise by approximately 50 degrees. It was possible to overlap and connect the structure of phospholamban[1-36] derived in the present study to the two transmembrane pentamer models proposed. In this way two models of the whole phospholamban in its pentameric form were generated. When our structure was connected to the leucine-isoleucine zipper model, the inner side of the cytoplasmic domain of the pentamer (where the helices face one another) was lined by polar residues (Gln23, Gln26, and Asn30), whereas the five Arg25 side chains were on the outer side. On the contrary, when our structure was connected to the other transmembrane model, in the inner side of the cytoplasmic domain of the pentamer, the five Arg25 residues formed a highly charged cluster.
Project description:Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure. In the present work, we investigate how this refolded state interacts with membrane interfaces in the early steps of T-domain's membrane association. Coarse-grained molecular dynamics simulations suggest two distinct membrane-bound conformations of the T-domain in the presence of bilayers composed of a mixture of zwitteronic and anionic phospholipids (POPC:POPG with a 1:3 molar ratio). Both membrane-bound conformations show a common near parallel orientation of hydrophobic helices TH8-TH9 relative to the membrane plane. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the N-terminal segment of the protein and the membrane interface. The second membrane-bound conformation is stabilized by hydrophobic interactions between protein residues and lipid acyl chains, which facilitate deeper protein insertion in the membrane interface. A theoretical estimate of a free energy of binding of a membrane-competent T-domain to the membrane is provided.
Project description:The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.
Project description:Complex I is the first and largest enzyme of the respiratory chain and has a central role in cellular energy production through the coupling of NADH:ubiquinone electron transfer to proton translocation. It is also implicated in many common human neurodegenerative diseases. Here, we report the first crystal structure of the entire, intact complex I (from Thermus thermophilus) at 3.3?Å resolution. The structure of the 536-kDa complex comprises 16 different subunits, with a total of 64 transmembrane helices and 9 iron-sulphur clusters. The core fold of subunit Nqo8 (ND1 in humans) is, unexpectedly, similar to a half-channel of the antiporter-like subunits. Small subunits nearby form a linked second half-channel, which completes the fourth proton-translocation pathway (present in addition to the channels in three antiporter-like subunits). The quinone-binding site is unusually long, narrow and enclosed. The quinone headgroup binds at the deep end of this chamber, near iron-sulphur cluster N2. Notably, the chamber is linked to the fourth channel by a 'funnel' of charged residues. The link continues over the entire membrane domain as a flexible central axis of charged and polar residues, and probably has a leading role in the propagation of conformational changes, aided by coupling elements. The structure suggests that a unique, out-of-the-membrane quinone-reaction chamber enables the redox energy to drive concerted long-range conformational changes in the four antiporter-like domains, resulting in translocation of four protons per cycle.