Andreas Kukol

Exploring models of the influenza A M2 channel: MD simulations in a phospholipid bilayer.

The M2 protein of influenza A virus forms homotetrameric helix bundles, which function as proton-selective channels. The native form of the protein is 97 residues long, although peptides representing the transmembrane section display ion channel activity, which (like the native channel) is blocked by the antiviral drug amantadine. As a small ion channel, M2 may provide useful insights into more complex channel systems. Models of tetrameric bundles of helices containing either 18 or 22 residues have been simulated while embedded in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine bilayer. Several different starting models have been used. These suggest that the simulation results, at least on a nanosecond time scale, are sensitive to the exact starting structure. Electrostatics calculations carried out on a ring of four ionizable aspartate residues at the N-terminal mouth of the channel suggest that at any one time, only one will be in a charged state. Helix bundle models were mostly stable over the duration of the simulation, and their helices remained tilted relative to the bilayer normal. The M2 helix bundles form closed channels that undergo breathing motions, alternating between a tetramer and a dimer-of-dimers structure. Under these conditions either the channel forms a pocket of trapped waters or it contains a column of waters broken predominantly at the C-terminal mouth of the pore. These waters exhibit restricted motion in the pore and are effectively frozen in a way similar to those seen in previous simulations of a proton channel formed by a four-helix bundle of a synthetic leucine-serine peptide (, Biophys. J. 77:2400-2410).

vpu Transmembrane Peptide Structure Obtained by Site-Specific Fourier Transform Infrared Dichroism and Global Molecular Dynamics Searching

The recently developed method of site-directed Fourier transform infrared dichroism for obtaining orientational constraints of oriented polymers is applied here to the transmembrane domain of the vpu protein from the human immunodeficiency virus type 1 (HIV-1). The infrared spectra of the 31-residue-long vpu peptide reconstituted in lipid vesicles reveal a predominantly alpha-helical structure. The infrared dichroism data of the (13)C-labeled peptide yielded a helix tilt beta = (6.5 +/- 1.7) degrees from the membrane normal. The rotational pitch angle omega, defined as zero for a residue located in the direction of the helix tilt, is omega = (283 +/- 11) degrees for the (13)C labels Val(13)/Val(20) and omega = (23 +/- 11) degrees for the (13)C labels Ala(14)/Val(21). A global molecular dynamics search protocol restraining the helix tilt to the experimental value was performed for oligomers of four, five, and six subunits. From 288 structures for each oligomer, a left-handed pentameric coiled coil was obtained, which best fits the experimental data. The structure reveals a pore occluded by Trp residues at the intracellular end of the transmembrane domain.

Site-specific examination of secondary structure and orientation determination in membrane proteins: The peptidic 13C18O group as a novel infrared probe

Detailed site‐specific information can be exceptionally useful in structural studies of macromolecules in general and proteins in particular. Such information is usually obtained from spectroscopic studies using a label/probe that can reflect on particular properties of the protein. A suitable probe must not modify the native properties of the protein, and should yield interpretable structural information, as is the case with isotopic labels used by Fourier transform infrared (FTIR) spectroscopy. In particular, 1‐13Cuf8feO labels have been shown to relay site‐specific secondary structure and orientational information, although limited to small peptides. The reason for this limitation is the high natural abundance of 13C and the lack of baseline resolution between the main amide I band and the isotope‐edited peak. Herein, we dramatically extend the utility of isotope edited FTIR spectroscopy to proteins of virtually any size through the use of a new 1‐13Cuf8fe18O label. The double‐isotope label virtually eliminates any contribution from natural abundance 13C. More importantly, the isotope‐edited peak is further red‐shifted (in accordance with ab initio Hartree–Fock calculations) and is now completely baseline resolved from the main amide I band. Taken together, this new label enables determination of site specific secondary structure and orientation in proteins of virtually any size. Even in small peptides 1‐13Cuf8fe18O is far preferable as a label in comparison to 1‐13Cuf8fe16O since it enables analysis without the need for any deconvolution or peak fitting procedures. Finally, the results obtained herein represent the first stage in the application of site‐directed dichroism to the structural elucidation of polytopic membrane proteins.

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.

The structure of the HIV-1 Vpu ion channel: modelling and simulation studies

Vpu is an 81 amino acid auxiliary protein in HIV-1 which exhibits channel activity. We used two homo-pentameric bundles with the helical transmembrane segments derived from FTIR spectroscopy in combination with a global molecular dynamics search protocol: (i) tryptophans (W) pointing into the pore, and (ii) W facing the lipids. Two equivalent bundles have been generated using a simulated annealing via a restrained molecular dynamics simulations (SA/MD) protocol. A fifth model was generated via SA/MD with all serines facing the pore. The latter model adopts a very stable structure during the 2 ns of simulation. The stability of the models with W facing the pore depends on the starting structure. A possible gating mechanism is outlined.

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 α‐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° ± 2° obtained by attenuated total reflection infrared spectroscopy. Thus, our data support the idea of ion channel formation by the PLM transmembrane domain.

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.

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 fromInfluenza 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 α-helical, and the helices are tilted by β = (14.6 ± 3.0)° from the membrane normal. The rotational pitch angle about the helix axis ω for the 1-13C-labeled residues Gly59 and Leu66 is ω = (218 ± 17)°, where ω 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 Ω = 16°. The putative transmembrane pore is occluded by the residue Met65. In addition hydrogen/deuterium exchange experiments show that the core is not accessible to water.

Mapping the energy surface of transmembrane helix-helix interactions.

Transmembrane helices are no longer believed to be just hydrophobic segments that exist solely to anchor proteins to a lipid bilayer, but rather they appear to have the capacity to specify function and structure. Specific interactions take place between hydrophobic segments within the lipid bilayer whereby subtle mutations that normally would be considered innocuous can result in dramatic structural differences. That such specificity takes place within the lipid bilayer implies that it may be possible to identify the most favorable interaction surface of transmembrane alpha-helices based on computational methods alone, as shown in this study. Herein, an attempt is made to map the energy surface of several transmembrane helix-helix interactions for several homo-oligomerizing proteins, where experimental data regarding their structure exist (glycophorin A, phospholamban, Influenza virus A M2, Influenza virus C CM2, and HIV vpu). It is shown that due to symmetry constraints in homo-oligomers the computational problem can be simplified. The results obtained are mostly consistent with known structural data and may additionally provide a view of possible alternate and intermediate configurations.

Conformational flexibility of the peptide hormone ghrelin in solution and lipid membrane bound: a molecular dynamics study.

Abstract Human ghrelin is a peptide hormone of 28 aminoacid residues, in which the Ser3 is modified by an octanoyl group. Ghrelin has a major role in the energy metabolism of the human body stimulating growth hormone release as well as food intake. Here we perform molecular dynamics simulations in explicit water and in a DMPC-lipid bilayer/water system in order to structurally characterize this highly flexible peptide and its lipid binding properties. We find a loop structure with residues Glu17 to Lys 20 in the bending region and a short α-helix from residues Pro7 to Glu13. The presence of a lipid membrane does not influence these structural features, but reduces the overall flexibility of the molecule as revealed by reduced root mean square fluctuations of the atom coordinates. The octanoyl-side chain does not insert into the lipid membrane but points into the water phase. The peptide binds to the lipid membrane with its bending region involving residues Arg15, Lys16, Glu17, and Ser18. The implications of these results for the binding pocket of the ghrelin receptor are discussed.