Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length

We examined the effect of the length of the hydrophobic core of Lys-flanked poly(Leu) peptides on their behavior when inserted into model membranes. Peptide structure and membrane location were assessed by the fluorescence emission lambdamax of a Trp residue in the center of the peptide sequence, the quenching of Trp fluorescence by nitroxide-labeled lipids (parallax analysis), and circular dichroism. Peptides in which the hydrophobic core varied in length from 11 to 23 residues were found to be largely alpha-helical when inserted into the bilayer. In dioleoylphosphatidylcholine (diC18:1PC) bilayers, a peptide with a 19-residue hydrophobic core exhibited highly blue-shifted fluorescence, an indication of Trp location in a nonpolar environment, and quenching localized the Trp to the bilayer center, an indication of transmembrane structure. A peptide with an 11-residue hydrophobic core exhibited emission that was red-shifted, suggesting a more polar Trp environment, and quenching showed the Trp was significantly displaced from the bilayer center, indicating that this peptide formed a nontransmembranous structure. A peptide with a 23-residue hydrophobic core gave somewhat red-shifted fluorescence, but quenching demonstrated the Trp was still close to the bilayer center, consistent with a transmembrane structure. Analogous behavior was observed when the behavior of individual peptides was examined in model membranes with various bilayer widths. Other experiments demonstrated that in diC18:1PC bilayers the dilution of the membrane concentration of the peptide with a 23-residue hydrophobic core resulted in a blue shift of fluorescence, suggesting the red-shifted fluorescence at higher peptide concentrations was due to helix oligomerization. The intermolecular self-quenching of rhodamine observed when the peptide was rhodamine-labeled, and the concentration dependence of self-quenching, supported this conclusion. These studies indicate that the mismatch between helix length and bilayer width can control membrane location, orientation, and helix-helix interactions, and thus may mismatch control both membrane protein folding and the interactions between membrane proteins.     

The double helix coiled coil structure of murein lipoprotein from Escherichia coli.

A rod-like structure is proposed for the murein lipoprotein of Escherichia coli, built of two parallel unbroken α-helices arranged in a coiled coil of the same type as in the muscle protein tropomyosin. The amino acid sequence has the required regular pattern of hydrophobic amino acids at intervals of three and four residues and the secondary structure predicted from the sequence is 80% helical. A space-filling model confirms that the coiled coil model is stereochemically reasonable, and energy calculations for a series of coils with different radii suggest that the best structure is one with the helix axes 8.25 Å apart. Energyrefined atomic co-ordinates have been calculated which show that the hydrophobic side-chains form a series of close-packed unstrained contacts between the two helices along the entire length of the sequence. On the basis of this study the hexagonal membrane pore model and the segmented helix model proposed by others seem unlikely. The coiled coil has a strongly hydrophilic outer surface, suggesting that the protein has a watery environment within the E. coli cell envelope and is not strictly a membrane protein. Probably only the fatty acid portion of the lipoprotein penetrates into the lipid region of the outer membrane, so that the protein may act as a tie or a spacer between the lipid and the murein wall.     

FTIR-Spectroscopy of multistranded coiled coil proteins.

Coiled coils of different order were investigated using infrared (IR) spectroscopy. Recently, we demonstrated that dimeric coiled coils display unique vibrational spectra with at least three separable bands instead of only one band of a classical alpha-helix in the amide I region.This was attributed to a distortion of the helical structure by the supercoil bending, giving rise to bands that are not observed in the undistorted helix. Here, we investigated coiled coils forming trimers, tetramers, and pentamers. These higher order coiled coils, in general, possess larger superhelical pitches, resulting in a smaller helical distortion. We found that all coiled coils studied, including the native dimeric GCN4 leucine zipper and its variants leading to parallel trimers and tetramers as well as the rod portions of fibritin (parallel trimer), alpha-actinin (antiparallel spectrin type trimer), and COMP (parallel pentamer), displayed the typical three band pattern of the coiled coil amide I spectra. However, the separation of these three bands and their positional deviation from the classical alpha-helical band position was correlated to the extent of the helical distortion as reflected by the pitch values of the supercoils. The most pronounced spectral anomaly was found for the tropomyosin dimer with a reported helical pitch of 137 A, whereas the smallest spectral distortion was found for the pentameric COMP complex and the tetrameric leucine zipper mutant, both with a pitch of about 205 A.     

A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions.

The leucine zipper is a dimeric coiled-coil protein structure composed of two amphipathic alpha-helices with the hydrophobic surfaces interacting to create the dimer interface. This structure has been found to mediate the dimerization of two abundant classes of DNA binding proteins: the bZIP and bHLH-Zip proteins. Several workers have reported that amino acids in the e and g positions of the coiled coil can modulate dimerization stability and specificity. Using the bZIP protein VBP as a host molecule, we report a thermodynamic scale (delta delta G) for 27 interhelical interactions in 35 proteins between amino acids in the g and the following e positions (g<==>e') of a leucine zipper coiled coil. We have examined the four commonly occurring amino acids in the e and g positions of bZIP proteins, lysine (K), arginine (R), glutamine (Q), glutamic acid (E), as well as the only other remaining charged amino acid aspartic acid (D), and finally alanine (A) as a reference amino acid. These results indicate that E<==>R is the most stable interhelical pair, being 0.35 kcal/mol more stable than E<==>K. A thermodynamic cycle analysis shows that the E<==>R pair is 1.33 kcal/mol more stable than A<==>A with -1.14 kcal/mol of coupling energy (delta delta Gint) coming from the interaction of E with R. The E<==>K coupling energy is only -0.14 kcal/mol. E interacts with more specificity than Q. The R<==>R pair is less stable than the K<==>K by 0.24 kcal/mol. R interacts with more specificity than K. Q forms more stable pairs with the basic amino acids K and R rather than with E. Changing amino acids in the e position to A creates bZIP proteins that form tetramers.     

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.     

The contribution of buried polar groups to the conformational stability of the GCN4 coiled coil

The dimeric interface of the leucine zipper coiled coil from GCN4 has been used to probe the contributions of hydrophobic and hydrogen bonding interactions to protein stability. We have determined the energetics of placing Ile or Asn residues at four buried positions in a two-stranded coiled coil. As expected, Ile is favored over Asn at these buried positions, but not as much as predicted by considering only the hydrophobic effect. It appears that interstrand hydrogen bonds form between the side-chains of the buried Asn residues and these contribute to the conformational stability of the coiled-coil peptides. However, these contributions are highly dependent on the locations of the Asn pairs. The effect of an Ile to Asn mutation is greatest at the N terminus of the peptide and decreases almost twofold as we move the substitution from the N to C-terminal heptads.     

Reinterpretation of GCN4-p1 folding kinetics: partial helix formation precedes dimerization in coiled coil folding.

The folding of coiled coil peptides has traditionally been interpreted in terms of native dimer and unfolded monomers. Calculations using AGADIR and experimental studies of fragments suggest that the monomers of the coiled coil peptide, GCN4-p1, contain significant residual helical structure. A simple model based on diffusion-collision theory predicts not only the measured folding rate within an order of magnitude, but also predicts remarkably well the effect of alanine to glyXcine mutations. We suggest that intrinsic helix stability is a major determinant of the folding rate of the GCN4 coiled coil.     

Phylogenetic occurrence of coiled coil proteins: Implications for tissue structure in metazoa via a coiled coil tissue matrix

We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained “extended” (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1–2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in Metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.     

Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production

F₁-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside the cylinder made of α₃β₃ subunits. The amino and carboxy termini of the γ-subunit form the axle, an α-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25-40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120° intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation.     

Extended sugar slide function for the periplasmic coiled coil domain of ScrY

Several bacterial outer membrane proteins have a periplasmic extension whose structure and function remain elusive. Here, the structure/function relationship of the N-terminal periplasmic domain of the sucrose-specific outer membrane channel ScrY was investigated. Circular dichroism and analytical centrifugation demonstrated that the N-terminal domain formed a parallel, three-stranded coiled coil. When this domain was fused to the maltose-specific channel LamB, permeation of maltooligosaccharides in liposomes increased with increasing sugar chain length whereas wild-type LamB showed the opposite effect. Current fluctuation analysis demonstrated increased off-rates for sugar transport through the fusion protein. Moreover, equilibrium dialysis showed an affinity of sucrose for the isolated N-terminal peptide. Together these results demonstrate a novel function for coiled coil domains, operating as an extended sugar slide.     

Engineering of the hydrophobic core of an alpha-helical coiled coil

The amino acid sequence that forms the alpha-helical coiled coil structure has a representative heptad repeat denoted by defgabc, according to their positions. Although the a and d positions are usually occupied by hydrophobic residues, hydrophilic residues at these positions sometimes play important roles in natural proteins. We have manipulated a few amino acids at the a and d positions of a de novo designed trimeric coiled coil to confer new functions to the peptides. The IZ peptide, which has four heptad repeats and forms a parallel triple-stranded coiled coil, has Ile at all of the a and d positions. We show three examples: (1) the substitution of one Ile at either the a or d position with Glu caused the peptide to become pH sensitive; (2) the metal ion induced alpha-helical bundles were formed by substitutions with two His residues at the d and a positions for a medium metal ion, and with one Cys residue at the a position for a soft metal ion; and (3) the AAB-type heterotrimeric alpha-helical bundle formation was accomplished by a combination of Ala and Trp residues at the a positions of different peptide chains. Furthermore, we applied these procedures to prepare an ABC-type heterotrimeric alpha-helical bundle and a metal ion-induced heterotrimeric alpha-helical bundle.     

A pivotal role of the coiled coil of Sir4

The C terminus of Sir4 forms a coiled-coil structure. The coiled-coil domain is responsible for the dimerization of Sir4 and contains the binding site of Sir3. Structural and biochemical analyses of the Sir4 coiled-coil domain provide important insights into the molecular mechanisms of Sir3-Sir4 interaction and the assembly of a ternary Sir2/Sir3/Sir4 complex that are essential for epigenetic control of gene expression in S. cerevisiae.