Structure of the influenza C virus CM2 protein transmembrane domain obtained by site-specific infrared dichroism and global molecular dynamics searching

The 115-residue protein CM2 from Influenza C virus has been recently characterized as a tetrameric integral membrane glycoprotein. Infrared spectroscopy and site-directed infrared dichroism were utilized here to determine its transmembrane structure. The transmembrane domain of CM2 is alpha-helical, and the helices are tilted by beta = (14.6 +/- 3.0) degrees from the membrane normal. The rotational pitch angle about the helix axis omega for the 1-(13)C-labeled residues Gly(59) and Leu(66) is omega = (218 +/- 17) degrees, where omega is defined as zero for a residue pointing in the direction of the helix tilt. A detailed structure was obtained from a global molecular dynamics search utilizing the orientational data as an energy refinement term. The structure consists of a left-handed coiled-coil with a helix crossing angle of Omega = 16 degrees. The putative transmembrane pore is occluded by the residue Met(65). In addition hydrogen/deuterium exchange experiments show that the core is not accessible to water.     

Experimentally based orientational refinement of membrane protein models: A structure for the Influenza A M2 H+ channel

The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure.     

The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics

ErbB-2 is a member of the family of epidermal growth factor receptors, which shows an oncogenic mutation in the rat gene neu, Val664Glu in the transmembrane domain that causes permanent dimerisation and subsequently leads to uncontrollable cell division and tumour formation. We have obtained the alpha-helical structure of the mutant transmembrane domain dimer experimentally with site-specific infrared dichroism (SSID) based on six transmembrane peptides with 13C18O carbonyl group-labelled residues. The derived orientational data indicate a local helix tilt ranging from 28(+/-6) degrees to 22(+/-4) degrees. Altogether using orientational constraints from SSID and experimental alpha-helical constraints while performing a systematic conformational search including molecular dynamics simulation in a lipid bilayer, we have obtained a unique experimentally defined atomic structure. The resulting structure consists of a right handed alpha-helical bundle with the residues Ile659, Val663, Leu667, Ile671, Val674 and Leu679 in the dimerisation interface. The right-handed bundle is in contrast to the left-handed structures obtained in previous modelling efforts. In order to facilitate tight helical packing, the spacious Glu664 residues do not interact directly but with water molecules that enter the bilayer.     

Infrared dichroism from the X-ray structure of bacteriorhodopsin

A detailed comparison with the three-dimensional protein structure provides a stringent test of the models and parameters commonly used in determining the orientation of the alpha-helices from the linear dichroism of the infrared amide bands, particularly in membranes. The order parameters of the amide vibrational transition moments are calculated for the transmembrane alpha-helices of bacteriorhodopsin by using the crystal structure determined at a resolution of 1.55 A (PDB accession number 1C3W). The dependence on the angle delta(M) that the transition moment makes with the peptide carbonyl bond is fit by the expression ((3)/(2)S(alpha) cos(2) alpha)cos(2)(delta(M) + beta) - 1/2S(alpha), where S(alpha) (0.91) is the order parameter of the alpha-helices, alpha (13 degrees ) is the angle that the peptide plane makes with the helix axis, and beta (11 degrees ) is the angle that the peptide carbonyl bond makes with the projection of the helix axis on the peptide plane. This result is fully consistent with the model of nested axial distributions commonly used in interpreting infrared linear dichroism of proteins. Comparison with experimental infrared dichroic ratios for bacteriorhodopsin yields values of Theta(A) = 33 +/- 1 degree, Theta(I) = 39.5 +/- 1 degree, and Theta(II) = 70 +/- 2 degrees for the orientation of the transition moments of the amide A, amide I, and amide II bands, respectively, relative to the helix axis. These estimates are close to those found for model alpha-helical polypeptides, indicating that side-chain heterogeneity and slight helix imperfections are unlikely to affect the reliability of infrared measurements of helix orientations.     

A structure for the trimeric MHC class II-associated invariant chain transmembrane domain

The major histocompatibility complex (MHC)-associated invariant chain (Ii) contains a single transmembrane domain that forms trimers. Ii is involved in the assembly of the MHC and antigen presentation, and is thus central to the function of the immune system. Here, we show by attenuated total reflectance, Fourier transform infrared (ATR-FTIR) spectroscopy that the transmembrane domain is alpha-helical and we provide a structural model of the transmembrane domain obtained by a combination of site-specific infrared dichroism and molecular dynamics (MD) simulations. This work resolves the backbone structure of a transmembrane peptide by multiple (13)C=(18)O labelling at ten different residues. A second purely computational approach, based on MD simulations of Ii transmembrane homologous sequences, yields a similar structure that is consistent with our experimental results. The structure presented forms a left-handed coiled coil with an average helix tilt of 13(+/-6) degrees; the residue Gln47 implicated in trimer formation forms strong interhelical contacts, Thr50 points to the inside of the trimeric coil and forms a network of hydrogen bonds.     

Use of a new label, (13)==(18)O, in the determination of a structural model of phospholamban in a lipid bilayer. Spatial restraints resolve the ambiguity arising from interpretations of mutagenesis data

A structural model of pentameric phospholamban (Plb) in a lipid bilayer has been derived using a combination of experimental data, obtained from ATR-FTIR site-directed dichroism, and the implementation of the resulting restraints during a molecular dynamics simulation. Plb (residues 24-52) has been synthesised incorporating a new label, 1-(13)C==(18)O, at residues 42 and 43. We have not only determined the tilt of the helices, 10(+/-6) degrees, but also the relative orientation of the transmembrane segments, with an omega angle of -32(+/-10) degrees for L42. This angle is taken as zero in the direction of the helix tilt. Plb is a simple test case where site-directed dichroism has been applied to resolve the indeterminacy arising from the mutagenesis data available. The results presented point specifically to a single structural model for Plb.     

Secondary structure, orientation, and oligomerization of phospholemman, a cardiac transmembrane protein

Human phospholemman (PLM) is a 72-residue protein, which is expressed at high density in the cardiac plasma membrane and in various other tissues. It forms ion channels selective for K+, Cl-, and taurine in lipid bilayers and colocalizes with the Na+/K+-ATPase and the Na+/Ca2+-exchanger, which may suggest a role in the regulation of cell volume. Here we present the first structural data based on synthetic peptides representing the transmembrane domain of PLM. Perfluoro-octaneoate-PAGE of reconstituted proteoliposomes containing PLM reveals a tetrameric homo-oligomerization. Infrared spectroscopy of proteoliposomes shows that the PLM peptide is completely alpha-helical, even beyond the hydrophobic core residues. Hydrogen/deuterium exchange experiments reveal that a core of 20-22 residues is not accessible to water, thus embedded in the lipid membrane. The maximum helix tilt is 17 degrees +/- 2 degrees obtained by attenuated total reflection infrared spectroscopy. Thus, our data support the idea of ion channel formation by the PLM transmembrane domain.     

Analysis of the segmental stability of helical peptides by isotope-edited infrared spectroscopy

Isotope-edited infrared spectroscopy has the ability to probe the segmental properties of long biopolymers. In this work, we have compared the infrared spectra of a model helical peptide ((12)C) Ac-W-(E-A-A-A-R)(6)-A-NH(2), described originally by Merutka et al. (Biochemistry 1991;30:4245-4248) and three derivatives that are (13)C labeled at the backbone carbonyl of alanines. The locations of six isotopically labeled alanines are at the N-terminal, C-terminal, and the middle two repeating units of the peptide. Variation in temperature from 1 degrees to 91 degrees C transformed the peptides from predominantly helical to predominantly disordered state. Amplitude and position of the infrared amide I' absorption bands from (12)C- and (13)C-labeled segments provided information about the helical content. Temperature dependence of infrared spectra was used to estimate segmental stability. As a control measure of overall peptide stability and helicity (independent of labeling), the temperature dependence of circular dichroism spectra in the far-UV range at identical conditions (temperature and solvent) as infrared spectra was measured. The results indicate that the central quarter of the 32 amino acids helix has the maximal helicity and stability. The midpoint of the melting curve of the central quarter of the helix is 5.4 +/- 0.8 degrees C higher than that of the termini. The N-terminal third of the helix is more helical and is 2.0 +/- 1.4 degrees C more stable than the C-terminus.     

C-deuterated alanine: a new label to study membrane protein structure using site-specific infrared dichroism.

The helix tilt and rotational orientation of the transmembrane segment of M2, a 97-residue protein from the Influenza A virus that forms H(+)-selective ion channels, have been determined by attenuated total reflection site-specific infrared dichroism using a novel labeling approach. Triple C-deuteration of the methyl group of alanine in the transmembrane domain of M2 was used, as such modification shifts the asymmetric and symmetric stretching vibrations of the methyl group to a transparent region of the infrared spectrum. Structural information can then be obtained from the dichroic ratios corresponding to these two vibrations. Two consecutive alanine residues were labeled to enhance signal intensity. The results obtained herein are entirely consistent with previous site-specific infrared dichroism and solid-state nuclear magnetic resonance experiments, validating C-deuterated alanine as an infrared structural probe that can be used in membrane proteins. This new label adds to the previously reported (13)C [double bond] (18)O and C-deuterated glycine as a tool to analyze the structure of simple transmembrane segments and will also increase the feasibility of the study of polytopic membrane proteins with site-specific infrared dichroism.     

Transmembrane four-helix bundle of influenza A M2 protein channel: structural implications from helix tilt and orientation.

The transmembrane portion of the M2 protein from the Influenza A virus has been studied in hydrated dimyristroylphosphotidylcholine lipid bilayers with solid-state NMR. Orientational constraints were obtained from isotopically labeled peptide samples mechanically aligned between thin glass plates. 15N chemical shifts from single site labeled samples constrain the molecular frame with respect to the magnetic field. When these constraints are applied to the peptide, modeled as a uniform alpha-helix, the tilt of the helix with respect to the bilayer normal was determined to be 33 degrees +/- 3 degrees. Furthermore, the orientation about the helix axis was also determined within an error of +/- 30 degrees. These results imply that the packing of this tetrameric protein is in a left-handed four-helix bundle. Only with such a large tilt angle are the hydrophilic residues aligned to the channel axis.     

Proline affects oligomerization of a coiled coil by inducing a kink in a long helix

The structural effect of a proline in a helix in trifluoroethanol (TFE)/water medium was examined on a 29-mer peptide and its proline analog derived from the leucine zipper (LZ)-like motif of gp41 (the transmembrane glycoprotein of HIV-1) by NMR and circular dichroism (CD) spectroscopies. Lower helical content was found for the proline mutant from the CD study. NMR data show that distortion of the helix by proline is local and occurs mainly on the N-terminal side of the substitution site. Molecular dynamics computation exhibits a bending of the helical axis of 30 degrees +/- 10 degrees, in agreement with X-ray diffraction results. Light-scattering experiments indicated that the average aggregation number of the proline-substituted mutant is substantially lower than that of the wild-type peptide. From the ratio of dissociation constants of the wild-type and the proline mutant peptides, the difference in free energy of trimeric formation is calculated to be 2.1 kcal/mol. Thermal stability, helicity, and the average aggregation number for the helix oligomers were found to be correlated. The structural alteration and the reduced coiled coil stability may account for the deficiency in the biological functions of the proline mutants of gp41 and in the inhibitory action of proline-substituted peptides. These effects may also be important in unraveling the roles played by proline in transmembrane proteins.     

Orientation of gramicidin A transmembrane channel. Infrared dichroism study of gramicidin in vesicles.

Polarized infrared spectroscopy has been used to investigate the orientation of gramicidin A incorporated in dimyristoylphosphatidylcholine liposomes. Dichroism measurements of the major lipid (C = O ester, PO2-, CH2) and peptide (amide A, I, II) bands were performed on liposomes (with or without gramicidin) oriented by air-drying. The mean orientation of the lipid groups and of the pi LD helix chain in the gramicidin has been determined. It can be inferred from infrared frequencies of gramicidin that the dominant conformation of the peptide in liposomes cannot be identified to the antiparallel double-helical dimer found in organic solution. No shift in lipid frequencies was observed upon incorporation of gramicidin in the liposomes. However, a slight reorganization of the lipid hydrocarbon chains which become oriented more closely to the normal to the bilayer is evidenced by a change in the dichroism of the CH2 vibrations. The infrared dichroism results of gramicidin imply a perpendicular orientation of the gramicidin transmembrane channel with the pi LD helix axis at less than 15 degrees with respect to the normal to the bilayer.     

The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.     

The chain length dependence of helix formation of the second transmembrane domain of a G protein-coupled receptor of Saccharomyces cerevisiae

The chain length dependence of helix formation of transmembrane peptides in lipids was investigated using fragments corresponding to the second transmembrane domain of the alpha-factor receptor from Saccharomyces cerevisiae. Seven peptides with chain lengths of 10 (M2-10; FKYLLSNYSS), 14 (M2-14), 18 (M2-18), 22 (M2-22), 26 (M2-26), 30 (M2-30) and 35 (M2-35; RSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSS) residues, respectively, were synthesized. CD spectra revealed that M2-10 was disordered, and all of the other peptides assumed partially alpha-helical secondary structures in 99% trifluoroethanol (TFE)/H(2)O. In 50% TFE/H(2)O, M2-30 assumed a beta-like structure. The other six peptides exhibited the same CD patterns as those found in 99% TFE/H(2)O. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1 ratio) vesicles, M2-22, M2-26, and M2-35 formed alpha-helical structures, whereas the other peptides formed beta-like structures. Fourier transform infrared spectroscopy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1) multilayers showed that M2-10, M2-14, M2-18, and M2-30 assumed beta-structures in this environment. Another homologous 30-residue peptide (M2-30B), missing residues SNYSS from the N terminus and extending to RSRKT on the C terminus, was helical in lipid bilayers, suggesting that residues at the termini of transmembrane domains influence their biophysical properties. Attenuated total reflection Fourier transform infrared spectroscopy revealed that M2-22, M2-26, M2-30B, and M2-35 were alpha-helical and oriented at angles of 12 degrees, 13 degrees, 36 degrees, and 34 degrees, respectively, with respect to the multilayer normal. This study showed that chain length must be taken into consideration when using peptides representing single transmembrane domains as surrogates for regions of an intact receptor. Furthermore, this work indicates that the tilt angle and conformation of transmembrane portions of G protein-coupled receptors may be estimated by detailed spectroscopic measurements of single transmembrane peptides.     

FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association

A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state F?rster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems. The applicability of the simulation-based fitting approach was verified on simulated FRET data and then applied to determine the structural properties of the well-known major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants Y24C, G38C, and T46C of this protein were produced and specifically labeled with the fluorescence label AEDANS. The energy transfer data from the natural tryptophan at position 26, which is used as a donor, to AEDANS were analyzed assuming a helix model for the transmembrane domain of the protein. As a result of the FRET data analysis, the topology and bilayer embedment of this domain were quantitatively characterized. The resulting tilt of the transmembrane helix of the protein is 18 +/- 2 degrees. The tryptophan is located at a distance of 8.5 +/- 0.5 A from the membrane center. No specific aggregation of the protein was found. The methodology developed here is not limited to M13 major coat protein and can be used in principle to study the bilayer embedment of any small protein with a single transmembrane domain.     

Disorder influence on linear dichroism analyses of smectic phases

Linear dichroism, the unequal absorption of parallel and perpendicular linear polarized light, is often used to determine the anisotropic ordering of rodlike polymers in a smectic phase, such as helices in a lipid bilayer. It is a measure of two properties of the sample: 1), orientation of the chromophore transition dipole moment (TDM) and 2), disorder. Since it is the orientation of the chromophore TDM that is needed for high resolution structural studies, it is imperative to either deconvolve sample disorder, or at a minimum, estimate its effect upon the calculated TDM orientation. Herein, a rigorous analysis of the effects of disorder is undertaken based on the recently developed Gaussian disorder model implemented in linear dichroism data. The calculation of both the rod tilt and rotational pitch angles as a function of the disorder and dichroism, yield the following conclusions: Disorders smaller than 5 degrees have a vanishingly small effect on the calculated polymer orientation, whereas values smaller than 10 degrees have a negligible effect on the calculated parameters. Disorders larger than 10 degrees have an appreciable effect on the calculated orientational parameters and as such must be estimated before any structural characterization. Finally the theory is tested on the HIV vpu transmembrane domain, employing experimental mosaicity measurements from x-ray reflectivity rocking scans and linear dichroism.     

Use of a single glycine residue to determine the tilt and orientation of a transmembrane helix. A new structural label for infrared spectroscopy

Site-directed dichroism is an emerging technique for the determination of membrane protein structure. However, due to a number of factors, among which is the high natural abundance of (13)C, the use of this technique has been restricted to the study of small peptides. We have overcome these problems through the use of a double C-deuterated glycine as a label. The modification of a single residue (Gly) in the transmembrane segment of M2, a protein from the Influenza A virus that forms H(+)-selective ion channels, has allowed us to determine its helix tilt and rotational orientation. Double C-deuteration shifts the antisymmetric and symmetric stretching vibrations of the CD(2) group in glycine to a transparent region of the infrared spectrum where the dichroic ratio of these bands can be measured. The two dichroisms, along with the helix amide I dichroic ratio, have been used to determine the helix tilt and rotational orientation of M2. The results are entirely consistent with previous site-directed dichroism and solid-state NMR experiments, validating C-deuterated glycine (GlyCD(2)) as a structural probe that can now be used in the study of polytopic membrane proteins.     

Helix-forming tendencies of amino acids depend on the restrictions of side-chain rotamer conformations: crystal structure of the tripeptide GAI in two crystalline forms.

In our attempts to design crystalline alpha-helical peptides, we synthesized and crystallized GAI (C11H21N3O4) in two crystal forms, GAI1 and GAI2. Form 1 (GAI1) Gly-L-Ala-L-Ile (C11H21N3O4.3H2O) crystals are monoclinic, space group P2(1) with a = 8.171(2), b = 6.072(4), c = 16.443(4) A, beta = 101.24(2) degrees, V = 800 A3, Dc = 1.300 g cm-3 and Z = 2, R = 0.081 for 482 reflections. Form 2 (GAI2) Gly-L-Ala-L-Ile (C11H21N3O4.1/2H2O) is triclinic, space group P1 with a = 5.830(1), b = 8.832(2), c = 15.008(2) A, alpha = 102.88(1), beta = 101.16(2), gamma = 70.72(2) degrees, V = 705 A3, Z = 2, Dc = 1.264 g cm-3, R = 0.04 for 2582 reflections. GAI1 is isomorphous with GAV and forms a helix, whereas GAI2 does not. In GAI1, the tripeptide molecule is held in a near helical conformation by a water molecule that bridges the NH3+ and COO- groups, and acts as the fourth residue needed to complete the turn by forming two hydrogen bonds. Two other water molecules form intermolecular hydrogen bonds in stabilizing the helical structure so that the end result is a column of molecules that looks like an incipient alpha-helix. GAI2 imitates a cyclic peptide and traps a water molecule. The conformation angles chi 11 and chi 12 for the side chain are (-63.7 degrees, 171.1 degrees) for the helical GAI1, and (-65.1 degrees, 58.6 degrees) and (-65.0 degrees, 58.9 degrees) for the two independent nonhelical molecules in GAI2; in GAI1, both the C gamma atoms point away from the helix, whereas in GAI2 the C gamma atom with the g+ conformation points inward to the helix and causes sterical interaction with atoms in the adjacent peptide plane. From these results, it is clear that the helix-forming tendencies of amino acids correlate with the restrictions of side-chain rotamer conformations. Both the peptide units in GAI1 are trans and show significant deviation from planarity [omega 1 = -168(1) degrees; omega 2 = -171(1) degrees] whereas both the peptide units in both the molecules A and B in GAI2 do not show significant deviation from planarity [omega 1 = 179.3(3) degrees; omega 2 = -179.3(3) degrees for molecule A and omega 1 = 179.5(3) degrees; omega 2 = -179.4(3) degrees for molecule B], indicating that the peptide planes in these incipient alpha-helical peptides are considerably bent.     

Convergence of experimental, computational and evolutionary approaches predicts the presence of a tetrameric form for CD3-zeta

Experimental results using multiple site-specific infrared dichroism have shown that, when reconstituted into lipid bilayers, the orientation of the transmembrane domain of CD3-zeta is not compatible with a dimeric right-handed model reported previously. This model, obtained using a computational approach that uses evolutionary data, is in agreement with mutagenesis data and homology modelling. This suggested that, in our experimental conditions, the oligomeric state of CD3-zeta may not be dimeric. We have explored this possibility by performing global searching molecular dynamics simulations assuming different homo-oligomeric sizes (from 2 to 6). In these simulations, the helix tilt was restrained to the average helix tilt obtained experimentally, 12 degrees. Only a left-handed tetrameric model was compatible with the experimentally observed tilt and rotational orientation of the helix, and was also the lowest-energy model amongst the candidate structures obtained. Furthermore, simulations performed using close homologues demonstrate that this model is compatible with evolutionary conservation data. Finally, the pattern of residue conservation in the zeta family of proteins strongly argues in favour of the presence of a left-handed hetero-oligomer with an orientation compatible with the tetramer we present. These results show that both the known dimeric and the so far undetected tetrameric form may be of functional importance in the cell.