Removing an interhelical salt bridge abolishes coiled-coil formation in a de novo designed peptide

Alpha-helical coiled coils represent a common protein oligomerization motif that are mainly stabilized by hydrophobic interactions occurring along their coiled-coil interface, the so-called hydrophobic seam. We have recently de novo designed and optimized a series of two-heptad repeat long coiled-coil peptides which are further stabilized by a complex network of inter- and intrahelical salt bridges. Here we have extended the de novo design of such two heptad-repeat long peptides by removing the central and most important g-e' Arg to Glu (g-e'RE) ionic interhelical interaction and replacing these residues by alanine residues. The effect of the missing interhelical ionic interaction on coiled-coil formation and stability has been analyzed by CD spectroscopy, analytical ultracentrifugation, and X-ray crystallography. We show that the peptide, while being highly alpha-helical, is no longer able to form a parallel coiled-coil structure but rather assumes an octameric globular helical assembly devoid of any coiled-coil interactions.     

Design of a minimal protein oligomerization domain by a structural approach

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.     

The coiled-coil trigger site of the rod domain of cortexillin I unveils a distinct network of interhelical and intrahelical salt bridges

BACKGROUND: The parallel two-stranded alpha-helical coiled coil is the most frequently encountered subunit-oligomerization motif in proteins. The simplicity and regularity of this motif have made it an attractive system to explore some of the fundamental principles of protein folding and stability and to test the principles of de novo design. RESULTS: The X-ray crystal structure of the 18-heptad-repeat alpha-helical coiled-coil domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum is a tightly packed parallel two-stranded alpha-helical coiled coil. It harbors a distinct 14-residue sequence motif that is essential for coiled-coil formation, and is a prerequisite for the assembly of cortexillin I. The atomic structure reveals novel types of ionic coiled-coil interactions. In particular, the structure shows that a characteristic interhelical and intrahelical salt-bridge pattern, in combination with the hydrophobic interactions occurring at the dimer interface, is the key structural feature of its coiled-coil trigger site. CONCLUSIONS: The knowledge gained from the structure could be used in the de novo design of alpha-helical coiled coils for applications such as two-stage drug targeting and delivery systems, and in the design of coiled coils as templates for combinatorial helical libraries in drug discovery and as synthetic carrier molecules.     

NMR solution structure of a highly stable de novo heterodimeric coiled-coil

The NMR solution structure of a highly stable coiled-coil IAAL-E3/K3 has been solved. The E3/K3 coiled-coil is a 42-residue de novo designed coiled-coil comprising three heptad repeats per subunit, stabilized by hydrophobic contacts within the core and electrostatic interactions at the interface crossing the hydrophobic core which direct heterodimer formation. This E3/K3 domain has previously been shown to have high alpha-helical content as well as possessing a low dissociation constant (70 nM). The E3/K3 structure is completely alpha-helical and is an archetypical coiled-coil in solution, as determined using a combination of (1)H-NOE and homology based structural restraints. This structure provides a structural framework for visualizing the important interactions for stability and specificity, which are key to protein engineering applications such as affinity purification and de novo design.     

Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper.

The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two-stranded alpha-helical coiled-coils as a model system. The native coiled-coil consists of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil structure at 20 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled-coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled-coils contained potential i to i' + 5 Glu-Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E2(20,22) coiled-coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' + 5 interchain Glu-Glu repulsions, which destabilize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized Glu side chains present at neutral pH. The protonated Glu residues increase the hydrophobicity of the coiled-coil interface leading to higher coiled-coil stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled-coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ionic nature and that Glu and Gln in the e and g positions of the heptad repeat have very similar effects on coiled-coil stability in the presence of GdnHCl.     

The two-stranded alpha-helical coiled-coil is an ideal model for studying protein stability and subunit interactions.

We have designed de novo a two-stranded alpha-helical coiled-coil which consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment. Their structure is stabilized by interchain hydrophobic interactions from hydrophobes at positions "a" and "d" of a repeating heptad sequence. The formation and stability of the coiled-coil is dependent on peptide concentration due to the monomer-dimer equilibrium. In contrast, that coiled-coil containing an inter-helical disulfide bond does not show any concentration dependence in the guanidine hydrochloride denaturation experiments as expected. Replacement of one large hydrophobic Leu residue in each chain with Ala significantly decreases coiled-coil stability in both the reduced and oxidized coiled-coils [decreases in transition midpoint of 1.6M (2.3-0.7) and 2.4M (5.3-2.9), respectively]. A large pH dependence on coiled-coil stability is observed over the pH range 4 to 7 (transition midpoints at pH 4, 5, 5.5, 6 and 7 were 3.8, 3.2, 2.0, 1.2 and 0.7M, respectively). The increasing stability with decreasing pH correlates with the protonation of the Glu acid side-chains and reduction of intrachain repulsions between Glu-Glu side-chains in positions i, i + 3 or i, i + 4 along each alpha-helix of the coiled-coil. In addition, coiled-coil stability increases with increasing ionic strength.     

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.     

Design, synthesis and structural characterization of model heterodimeric coiled-coil proteins.

We report the design and synthesis of model heterodimeric coiled-coil proteins and the packing contribution of interchain hetero-hydrophobic side-chains to coiled-coil stability. The heterodimeric coiled-coils are obtained by oxidizing two 35-residue polypeptide chains, each containing a cysteine residue at position 2 and differing in amino acid sequences in the hydrophobic positions ("a" and "d") responsible for the formation and stabilization of the coiled-coil. In each peptide, a single Ala residue was substituted for Leu at position "a" or "d". The formation and stability of heterodimeric coiled-coils were investigated by circular dichroism studies in the presence and absence of guanidine hydrochloride and compared to the corresponding homodimeric coiled-coils. The coiled-coil proteins with an Ala substitution at position "a" were less stable than those with an Ala substitution at position "d" in both the homodimeric (Ala-Ala interchain interactions) and heterodimeric (Leu-Ala interchain interactions ) coiled-coils. The 70-residue disulfide bridged peptides (homo- and heterodimeric coiled-coils) can be readily separated by reversed-phase chromatography (RPC) even though they have identical amino acid compositions as well as in the hydrophobic "a" and "d" positions. The elution of the 70-residue peptides prior to their corresponding 35-residue monomers suggests that these proteins are retaining a large portion of their coiled-coil structure during RPC at pH2 and their retention behavior correlates with protein stability.     

The role of position a in determining the stability and oligomerization state of alpha-helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled-coil protein in determining coiled-coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three-stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys-Gly-Gly linker, unfolding occurred from the same two-stranded monomeric oligomerization state for all of the analogs. Denaturation from a two-stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled-coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed-phase high-performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled-coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high-performance size-exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 microM under benign buffer conditions. The Leu-, Tyr-, Gln-, and His-substituted analogs were found to be exclusively three-stranded trimers, while the Asn-, Lys-, Orn-, Arg-, and Trp-substituted analogs formed exclusively two-stranded monomers. Modeling results for the Leu-substituted analog showed that a three-stranded oligomerization state is preferred due to increased side-chain burial, while a two-stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three-stranded state.     

A single amino acid can switch the oligomerization state of the alpha-helical coiled-coil domain of cartilage matrix protein.

We have studied the oligomerization of an alpha-helical coiled-coil using as an example a peptide corresponding to the C-terminal domain of cartilage matrix protein. By replacing one arginine residue, which forms an interchain ionic interaction with a glutamic acid residue, with glutamine, we found that this peptide assembles into a homotetramer at neutral pH in contrast to the native molecule which forms homotrimers. At acidic and basic pH, however, we again observed the trimer conformation. Another arginine, which is probably involved in an intrachain salt bridge, has no effect on the assembly. Our data demonstrate that besides the specific distribution of hydrophobic residues, interchain ionic interactions can be crucial in modulating the association behavior of alpha-helical coiled-coil domains.     

Side chain-backbone hydrogen bonding contributes to helix stability in peptides derived from an alpha-helical region of carboxypeptidase A

Recently, Presta and Rose proposed that a necessary condition for helix formation is the presence of residues at the N- and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.     

Role of hydrophobic clusters in the stability of alpha-helical coiled coils and their conversion to amyloid-like beta-sheets

We designed a library of short peptides using standard rules for coiled-coil assembly. Depending on the composition of amino acids in the non-interacting region of the coiled coil (positions b, c, and f) these peptides are able to convert from alpha-helical to beta-sheet secondary structure. This type of transition is observed in amyloid-like proteins and is a key feature associated with many types of neurodegenerative diseases. Studies on peptides that are 14 amino acids in length indicated that positioning hydrophobic amino acids at an f position within a heptad repeat accelerated the rate of conformational conversion as compared to that at a c position. We believe that this occurs because of the formation of a hydrophobic pocket that preferentially stabilizes beta-sheets over alpha-helices. This effect was also observed in longer 21 amino acid peptides. Our study shows that the relative rates of structural conversion correlate with the formation of a continuous three-amino-acid hydrophobic patch consisting of amino acids in the d, f, and a positions and not on the secondary structure propensities of the individual amino acids. The sequence-structure relationship observed in this study will be used to help understand the mechanism of amyloid fiber formation and design future coiled-coil and beta-sheet-forming peptide systems.     

Design and characterization of a model αβ peptide

CD and nmr spectroscopy were used to compare the conformational properties of two related peptides. One of the peptides, Model AB, was designed to adopt a helix-turn-extended strand (αβ) tertiary structure in water that might be stabilized by hydrophobic interactions between two leucine residues in the amino-terminal segment and two methionine residues in the carboxyl terminal segment. The other peptide, AB Helix, has the same amino acid sequence as Model AB except that it lacks the-Pro-Met-Thr-Met-Thr-Gly segment at the carboxyl-terminus. Although the carboxyl-terminal segment of Model AB was found to be unstructured, its presence increases the number of residues in a helical conformation, shifts the pKas of three ionizable side chains by 1 pH unit or more compared to an unstructured peptide, stabilizes the peptide as a monomer in high concentrations of ammonium sulfate, increases the conformational stability of residues at the terminal ends of the helix, and results in many slowly exchanging amide protons throughout the entire backbone of the peptide. These results suggest that interactions between adjacent segments in a small peptide can have significant structure organizing effects. Similar kinds of interactions may be important in determining the structure of early intermediates in protein folding and may be useful in the de novo design of independently folding peptides. © 1995 John Wiley & Sons, Inc.     

The hydrophobic pocket contributes to the structural stability of the N-terminal coiled coil of HIV gp41 but is not required for six-helix bundle formation

In models of HIV fusion, the glycoprotein gp41 is thought to form a six-helix bundle during viral fusion with the target cell. This bundle is comprised of three helical regions (from the heptad repeat 2, or HR2, region of gp41) bound to an inner, trimeric, coiled-coil core (from the HR1 region). Although much has been learned about the structure and thermodynamics of this complex, the energetics of the isolated HR1 self-associated oligomer remain largely unknown. By systematically studying self-association through a series of truncations based on a 51-mer HR1 peptide (T865), we have identified amino acid segments which contribute significantly to the stability of the oligomeric HR1 complex. Biophysical characterization of C-terminal truncations of T865 identifies a 10-15-amino acid region that is essential for HR1 oligomerization. This region coincides with a hydrophobic pocket that provides important contacts for the interaction of HR2 helices. Complete removal of this pocket abolishes HR1 oligomerization. Despite the dramatic reduction in stability, the monomeric HR1 peptides are still able to form stable six-helix bundles in the presence of HR2 peptides. Truncations on the N-terminal side of T865 have little effect on oligomerization but significantly reduce the stability of the HR1-HR2 six-helix bundle. Unlike the HR2 binding site, which extends along a hydrophobic groove on the HR1 oligomer, the residues that are critical for HR1 oligomerization are concentrated in a 10-15-amino acid region. These results demonstrate that there are localizations of binding energy, or "hot spots", in the self-association of peptides derived from the HR1 region of gp41.     

Probing the initial stage of aggregation of the Abeta(10-35)-protein: assessing the propensity for peptide dimerization

Characterization of the early stages of peptide aggregation is of fundamental importance in elucidating the mechanism of the formation of deposits associated with amyloid disease. The initial step in the pathway of aggregation of the Abeta-protein, whose monomeric NMR structure is known, was studied through the simulation of the structure and stability of the peptide dimer in aqueous solution. A protocol based on shape complementarity was used to generate an assortment of possible dimer structures. The structures generated based on shape complementarity were evaluated using rapidly computed estimates of the desolvation and electrostatic interaction energies to identify a putative stable dimer structure. The potential of mean force associated with the dimerization of the peptides in aqueous solution was computed for both the hydrophobic and the electrostatic driven forces using umbrella sampling and classical molecular dynamics simulation at constant temperature and pressure with explicit solvent and periodic boundary conditions. The comparison of the two free energy profiles suggests that the structure of the peptide dimer is determined by the favorable desolvation of the hydrophobic residues at the interface. Molecular dynamics trajectories originating from two putative dimer structures indicate that the peptide dimer is stabilized primarily through hydrophobic interactions, while the conformations of the peptide monomers undergo substantial structural reorganization in the dimerization process. The finding that the phi-dimer may constitute the ensemble of stable Abeta(10-35) dimer has important implications for fibril formation. In particular, the expulsion of water molecules at the interface might be a key event, just as in the oligomerization of Abeta(16-22) fragments. We conjecture that events prior to the nucleation process themselves might involve crossing free energy barriers which depend on the peptide-peptide and peptide-water interactions. Consistent with existing experimental studies, the peptides within the ensemble of aggregated states show no signs of formation of secondary structure.     

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.     

Reversible assembly of helical filaments by de novo designed minimalist peptides

We have designed a series of 15 short, helical de novo peptides consisting of lysine, isoleucine, and alanine. We have termed this the KIA series. These peptides differ only in their hydrophobic interface, and thus their self-association is largely a consequence of hydrophobic interactions. One of these peptides, KIA13, forms insoluble helical fibers at specific NaCl concentrations. We have used CD spectroscopy, turbidity assays, and in situ tapping mode atomic force microscopy to characterize the reversible assembly pathway for this peptide. It is unfolded at low NaCl concentration, and forms helical, soluble fibers resembling a coiled-coil conformation at intermediate NaCl concentrations, and rope-like insoluble fibers at high NaCl concentrations. Reducing the NaCl concentration completely reverses this process. Another peptide from the KIA series specifically inhibits the formation of the insoluble KIA13 fibers, and reverses the process to some extent. This work sheds light onto protein fibrillogenesis and offers intriguing possibilities for the use of these types of peptides in drug delivery and biomaterials applications.     

Effect of chain length on coiled-coil stability: decreasing stability with increasing chain length

The de novo design and biophysical characterization of three series of two-stranded alpha-helical coiled coils with different chain lengths are described. Our goal was to examine how increasing chain length would affect protein folding and stability when one or more heptad repeat(s) of K-A-E-A-L-E-G (gabcdef) was inserted into the central region of different coiled-coil host proteins. This heptad was designed to maintain the continuous 3-4 hydrophobic repeat of the coiled-coil host and introduce an Ala and Leu residue in the hydrophobic core at the a and d position, respectively, and a pair of stabilizing interchain ionic i to i' + 5 (g to e') interactions per heptad inserted. The secondary structures of the three series of disulfide-bridged polypeptides were studied by CD spectroscopy and their stabilities determined by chemical and thermal denaturation. The results showed that successive insertions of this heptad systematically decreased the stability of all the coiled coils studied regardless of the overall initial stability of the host coiled coil. These observations are in contrast to the generally accepted implication that the folding and stability of coiled coils are enhanced with increasing chain length. Our results imply that, in these examples where an Ala and Leu hydrophobic residue were introduced into the coiled-coil core per inserted heptad, there was still insufficient stability to overcome unfavorable entropy associated with chain length extension, even though the inserted heptad contained the most stabilizing hydrophobic residue (Leu) at position d and stabilizing ionic attractions.     

Clustering of large hydrophobes in the hydrophobic core of two-stranded alpha-helical coiled-coils controls protein folding and stability

The de novo design and biophysical characterization of two 60-residue peptides that dimerize to fold as parallel coiled-coils with different hydrophobic core clustering is described. Our goal was to investigate whether designing coiled-coils with identical hydrophobicity but with different hydrophobic clustering of non-polar core residues (each contained 6 Leu, 3 Ile, and 7 Ala residues in the hydrophobic core) would affect helical content and protein stability. The disulfide-bridged P3 and P2 differed dramatically in alpha-helical structure in benign conditions. P3 with three hydrophobic clusters was 98% alpha-helical, whereas P2 was only 65% alpha-helical. The stability profiles of these two analogs were compared, and the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence by circular dichroism spectroscopy and confirmed by differential scanning calorimetry. The results showed that P3 assembled into a stable alpha-helical two-stranded coiled-coil and exhibited a native protein-like cooperative two-state transition in thermal melting, chemical denaturation, and calorimetry experiments. Although both peptides have identical inherent hydrophobicity (the hydrophobic burial of identical non-polar residues in equivalent heptad coiled-coil positions), we found that the context dependence of an additional hydrophobic cluster dramatically increased stability of P3 (Delta Tm approximately equal to 18 degrees C and Delta[urea](1/2) approximately equal to 1.5 M) as compared with P2. These results suggested that hydrophobic clustering significantly stabilized the coiled-coil structure and may explain how long fibrous proteins like tropomyosin maintain chain integrity while accommodating polar or charged residues in regions of the protein hydrophobic core.