X-ray structure of a water-soluble analog of the membrane protein phospholamban: sequence determinants defining the topology of tetrameric and pentameric coiled coils

Phospholamban (PLB) is a pentameric transmembrane protein that regulates the Ca(2+)-dependent ATPase SERCA2a in sarcoplasmic reticulum membranes. We previously described the computational design of a water-soluble variant of phospholamban, WSPLB, which reproduced many of the structural and functional properties of the native membrane-soluble protein. While the full-length WSPLB forms a pentamer in solution, a truncated variant forms very stable tetramers. To obtain insight into the tetramer-pentamer cytoplasmic switch, we solved the crystal structure of the truncated construct, WSPLB 21-52. This peptide has a heptad sequence repeat with Leu residues at a- and Ile at d-positions from residues 31-52. The crystal structure revealed that WSPLB 21-52 adopted an antiparallel tetrameric coiled coil. This topology contrasts with the parallel topology of an analogue of the coiled-coil of GCN4 with the same Leu(a) Ile(d) repeat. Analysis of these structures revealed how the nature of the partially exposed residues at e- and g-positions influence the topology formed by the bundle. We also constructed a model for the pentameric form of PLB using the coiled-coil parameters derived from a single monomer in the tetrameric structure. This model suggests that both buried and interfacial hydrogen bonds are important for stabilizing the parallel pentamer.     

Conformational specificity of the lac repressor coiled-coil tetramerization domain

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.     

Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant

The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel double-stranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.     

Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.     

Coiled-Coil Formation on Lipid Bilayers—Implications for Docking and?Fusion Efficiency

Coiled-coil formation of four different oligopeptides was characterized in solution, on hydrogels, and on membranes by employing circular dichroism spectroscopy, surface plasmon resonance spectroscopy, attenuated total reflection infrared spectroscopy, and ellipsometry. Peptide sequences rich in either glutamic acid (E: E3Cys, i-E3Cys) or lysine (K: K3Cys, i-K3Cys) were used to represent minimal mimics of eukaryotic SNARE motifs. Half of the peptides were synthesized in reverse sequence, so that parallel and antiparallel heptad coiled-coil structures were formed. Either E-peptides or K-peptides were attached covalently to phospholipid anchors via maleimide chemistry, and served as receptors for the recognition of the corresponding binding partners added to solution. Attenuated total reflection infrared spectroscopy of single bilayers confirmed the formation of coiled-coil complexes at the membrane interface. Coiled-coil formation in solution, as compared with association at the membrane surface, displays considerably larger binding constants that are largely attributed to loss of translational entropy at the interface. Finally, the fusogenicity of the various coiled-coil motifs was explored, and the results provide clear evidence that hemifusion followed by full fusion requires a parallel orientation of α-helices, whereas antiparallel oriented coiled-coil motifs display only docking.     

The role of the N-terminal heptad repeat of HIV-1 in the actual lipid mixing step as revealed by its substitution with distant coiled coils

The gp41 glycoprotein of HIV-1 is considered to be responsible for the actual fusion process between the virus and the host membranes. According to a prevailing model, gp41 trimer organization, directed by the N-terminal coiled-coil region (NHR), is essential for steps involved in the actual merging of viral and cellular membranes. This study addresses a major question: Is the specific sequence of the NHR obligatory for the fusion process, or can it be replaced by distant coiled coils that form different oligomeric states in solution? For this purpose we synthesized three known GCN4 coiled-coil mutants that oligomerize in solution into either dimers, trimers, or tetramers. These peptides were chemically ligated to the fusion peptide thereby creating three chimera peptides with different oligomeric tendencies in solution. These peptides were investigated, together with the 70-mer wild-type peptide (N70), regarding their structure in solution and membrane by using circular dichroism (CD) and FTIR spectroscopies, their ability to induce vesicle fusion, and their ability to bind phospholipid membranes by using surface plasmon resonance (SPR). Our results suggest that local assembly of fusion peptides, facilitated by coiled-coil oligomers, increases lipid mixing ability, probably by facilitating stronger binding of the fusion peptides to the opposing membrane as revealed by SPR. However, N70 is significantly more active than the other chimeras. Overall, the data indicate a correlation between the distinct conformation of N70 in solution and in membranes and its enhanced lipid mixing relative to the GCN4 chimeras.     

Pore Structure and Synergy in Antimicrobial Peptides of the Magainin Family

Magainin 2 and PGLa are among the best-studied cationic antimicrobial peptides. They bind preferentially to negatively charged membranes and apparently cause their disruption by the formation of transmembrane pores, whose detailed structure is still unclear. Here we report the results of 5–9 μs all-atom molecular dynamics simulations starting from tetrameric transmembrane helical bundles of these two peptides, as well as their stoichiometric mixture, and the analog MG-H2 in DMPC or 3:1 DMPC/DMPG membranes. The simulations produce pore structures that appear converged, although some effect of the starting peptide arrangement (parallel vs. antiparallel) is still observed on this timescale. The peptides remain mostly helical and adopt tilted orientations. The calculated tilt angles for PGLa are in excellent agreement with recent solid state NMR experiments. The antiparallel dimer structure in the magainin 2 simulations resembles previously determined NMR and crystal structures. More transmembrane orientations and a larger and more ordered pore are seen in the 1:1 heterotetramer with an antiparallel helix arrangement. Insights into the mechanism of synergy between these two peptides are obtained via implicit solvent modeling of homo- and heterodimers and analysis of interactions in the atomistic simulations. This analysis suggests stronger pairwise interactions in the heterodimer than in the two homodimers.     

Unique stabilizing interactions identified in the two-stranded alpha-helical coiled-coil: crystal structure of a cortexillin I/GCN4 hybrid coiled-coil peptide

We determined the 1.17 A resolution X-ray crystal structure of a hybrid peptide based on sequences from coiled-coil regions of the proteins GCN4 and cortexillin I. The peptide forms a parallel homodimeric coiled-coil, with C(alpha) backbone geometry similar to GCN4 (rmsd value 0.71 A). Three stabilizing interactions have been identified: a unique hydrogen bonding-electrostatic network not previously observed in coiled-coils, and two other hydrophobic interactions involving leucine residues at positions e and g from both g-a' and d-e' interchain interactions with the hydrophobic core. This is also the first report of the quantitative significance of these interactions. The GCN4/cortexillin hybrid surprisingly has two interchain Glu-Lys' ion pairs that form a hydrogen bonding network with the Asn residues in the core. This network, which was not observed for the reversed Lys-Glu' pair in GCN4, increases the combined stability contribution of each Glu-Lys' salt bridge across the central Asn15-Asn15' core to approximately 0.7 kcal/mole, compared to approximately 0.4 kcal mole(-1) from a Glu-Lys' salt bridge on its own. In addition to electrostatic and hydrogen bonding stabilization of the coiled-coil, individual leucine residues at positions e and g in the hybrid peptide also contribute to stability by 0.7 kcal/mole relative to alanine. These interactions are of critical importance to understanding the stability requirements for coiled-coil folding and in modulating the stability of de novo designed macromolecules containing this motif.     

Pitch diversity in α-helical coiled coils

Two complementary methods for measuring local pitch based on heptad position in α-helical coiled coils are described and applied to six crystal structures. The results reveal a diversity of pitch values: two-stranded coiled coils appear to have pitch values near 150 Å the values for three- and four-stranded coiled coils range closer to 200 Å. The methods also provide a rapid and sensitive gauge of local coiled-coil conformation. Polar or charged residues in the apolar interface between coiled-coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled-coil stability. Moreover, the identification of a skip residue (heptad frame shift) in the hemaglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes. These results on relatively short coiled-coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established. We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high-resolution crystal structure—a result not readily discerned by direct comparison of atomic coordinates. Taken together, these methods reveal specific aspects of coiled-coil structure which may escape detection by global analyses of pitch. © 1993 Wiley-Liss, Inc.     

Stabilization of coiled-coil peptide domains by introduction of trifluoroleucine

Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13 degrees C at 30 microM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5-1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.     

X-ray crystal structure of a TRPM assembly domain reveals an antiparallel four-stranded coiled-coil

Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.     

Detection of alpha-helical coiled-coil dimer formation by spin-labeled synthetic peptides: a model parallel coiled-coil peptide and the antiparallel coiled coil formed by a replica of the ProP C-terminus

Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide. The model peptide is known to form a stable, parallel homodimeric coiled coil, which is partially destabilized by Cys substitutions at heptad a and d positions (peptides C30a and C33d). The ProP peptide, a 30-residue synthetic peptide, corresponds to residues 468-497 of osmoregulatory transporter ProP from Escherichia coli. It forms a relatively unstable, homodimeric coiled coil that is predicted to be antiparallel in orientation. Cys was introduced in heptad g positions of the ProP peptide, near the N-terminus (K473C, creating peptide C473g) or closer to the center of the sequence (E480C, creating peptide C480g). In contrast to the destabilizing effect of Cys substitution at the core heptad a or d positions of model peptides C30a and C33d, circular dichroism spectroscopy showed that Cys substitutions at the heptad g positions of the ProP peptide had little or no effect on coiled-coil stability. Thermal denaturation analysis showed that spin labeling increased the stability of the coiled coil for all peptides. Strong exchange broadening was detected for both C30a and C33d, in agreement with a parallel structure. EPR spectra of C480g had a large hyperfine splitting of about 90 G, indicative of strong dipole-dipole interactions and a distance between spin-labeled residues of less than 9 A. Spin-spin interactions were much weaker for C473g. These results supported the hypothesis that the ProP peptide primarily formed an antiparallel coiled coil, since formation of a parallel dimer should result in similar spin-spin interactions for the spin-labeled Cys at both sites.     

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.     

Orientational Ambiguity in Septin Coiled Coils and its Structural Basis

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function.     

Conformational transition between four and five-stranded phenylalanine zippers determined by a local packing interaction

Alpha-helical coiled coils play a crucial role in mediating specific protein-protein interactions. However, the rules and mechanisms that govern helix-helix association in coiled coils remain incompletely understood. Here we have engineered a seven heptad "Phe-zipper" protein (Phe-14) with phenylalanine residues at all 14 hydrophobic a and d positions, and generated a further variant (Phe-14(M)) in which a single core Phe residue is substituted with Met. Phe-14 forms a discrete alpha-helical pentamer in aqueous solution, while Phe-14(M) folds into a tetrameric helical structure. X-ray crystal structures reveal that in both the tetramer and the pentamer the a and d side-chains interlock in a classical knobs-into-holes packing to produce parallel coiled-coil structures enclosing large tubular cavities. However, the presence of the Met residue in the apolar interface of the tetramer markedly alters its local coiled-coil conformation and superhelical geometry. Thus, short-range interactions involving the Met side-chain serve to preferentially select for tetramer formation, either by inhibiting a nucleation step essential for pentamer folding or by abrogating an intermediate required to form the pentamer. Although specific trigger sequences have not been clearly identified in dimeric coiled coils, higher-order coiled coils, as well as other oligomeric multi-protein complexes, may require such sequences to nucleate and direct their assembly.