Heterocyclic Peptide Backbone Modifications in an α-Helical Coiled Coil

Among a number of elegant biomimetic approaches that have been developed for the modification of the peptide backbone¹, only a few have been applied to protein structures.² Here, we report high resolution structural consequences of amide backbone replacements in the context of a folded peptide architecture. We show that a triazole ε²-amino acid (Figure 1a)³ can be used as a dipeptide surrogate in α-helical coiled coils and report on the effects of the substitution on the thermodynamic stability, helical secondary structure, and the four helix bundle quaternary organization.

Figure 1.

(a) A native dipeptide and the L-leucine derived triazole-ε²- amino acid incorporated as a replacement; (b) Sequences for GCN4-pLI and modified peptides 1-3; X denotes incorporation of the ε²-residue.

The reactivity and functional group tolerance of Cu(I) catalyzed 1,3-dipolar cycloaddition between azides and alkynes to give 1,2,3-triazoles⁴ have led to the increasing use of this reaction in bioconjugate chemistry.⁵ We reported recently the use of triazole ε²-amino acids as a dipeptide replacement in the design of selfassembling peptide nanotubes.⁶ We hypothesized that the structural and functional features of the triazole ε²-amino acids could also be potentially useful in the context peptide and protein secondary structures. The backbone of the ε-amino acid is one atom longer as compared to that of a native dipeptide (Figure 1a), leading to a calculated increase in C_α-C_αspacing of 1.1 Å.⁷ In addition to the amide NH and carbonyl groups flanking the residue, the triazole ring possesses two nitrogen atoms (N² and N³) that might act as hydrogen bond acceptors. Furthermore, the triazole ring has a large dipole that could align with that of the other amides in a given peptide secondary structure.⁸

We selected the pLI mutant of the α-helical coiled coil GCN4 in order to test the utility of the ε²-amino acid substitution in the context of a peptide with well defined secondary and quaternary structure in solution and the solid state.⁹ In coiled coils, interhelical interactions between buried hydrophobic residues as well as exposed hydrophilic side chains lead to robust peptide self-assembly into α-helical bundles. We replaced dipeptides K₈L₉, K₁₅L₁₆, and E₂₂L₂₃ in the pLI-GCN4 sequence with an Lleucine derived triazole ε-amino acid to give sequences 1, 2, and 3, respectively (Figure 1b). In each case, the isobutyl side chain of the ε-residue was predicted to replace a leucine residue in the hydrophobic core of the native 4-helix bundle. The amino acid employed was synthesized in two steps from L-leucine and used in standard solid phase peptide synthesis conditions.

Circular dichroism (CD) spectra of peptides 1-3 (50 μM in 10 mM MOPS, pH 7.0) showed minima at 208 nm and 222 nm, characteristic of α-helical secondary structure (Figure S2). Thermal denaturation, monitoring the molar ellipticity at 222 nm, indicated broad two-state transitions for peptides 2 and 3, while peptide 1, similar to the parent pLI-GCN4 sequence, was considerably more stable and did not fully denature up to 96°C.⁹ Gel permeation chromatography indicated that peptides 1 and 3 adopt tetrameric oligomerization states in solution, while peptide 2 appears to exist primarily as a dimeric assembly (Table 1). These studies indicate that the modified peptides retain much of the native α-helical character, but that the position of the ε²- amino acid substitution differentially influences thermodynamic stability.

Table 1.

Biophysical Data and PDB IDs for Peptides 1-3^a

peptide	[θ]222 (deg cm2 dmol−1)b	Tm(°C)	Naggc	PDB ID
GCN4-pLId	−30,600	>96	4	1GCL
1	peptide 28,000	>96	4	1U9G
2	peptide 13,100	36	2	1U9F
3	peptide 21,000	61	4	1U9H

^asee Supporting Information for experimental details and full spectra;

^bat 4°C;

^capparent aggregation state in solution as determined by gel permeation chromatography;

^dvalues and structure from reference.⁹

We employed X-ray crystallography to ascertain the structural consequences of the ε²-amino acid substitutions in the context of the helical coiled coil architecture. Crystal structures were obtained for peptides 1-3 at 2.2 Å resolution. Although each peptide crystallized in a different unit cell and space group, all three exhibited the parallel tetrameric coiled coil structure of the parent pLI-GCN4 sequence with a crystallographic 2-fold symmetry axis along the center of each bundle (Figure 2, S3). In peptides 2 and 3, a complete well resolved structure similar to the parent pLI-GCN4 was obtained. However, in the case of 1, the electron density for portion of the peptide preceding the triazole ε-amino substitution (residues 1-8) was not observed, presumably due to chain disorder in the crystal (Figure S3).

Figure 2.

Schematic representation of the crystal structure of peptides 2 (a) and 3 (b) with atomic positions shown for the triazole residues. Each four-helix bundle superposes a crystallographic 2-fold axis and unique chains in each structure are indicated by different colors.10

The crystal structure of 2 is similar to that of the parent pLI with the hydrophobic side chain of the ε-residue projecting toward the core of the bundle (Figure S4). Notably, the ε-residue in each chain fully participates in α-helical backbone hydrogen bonding (Figure 3a). The N² of the triazole accepts a hydrogen bond from the amide NH of Ile₁₈ and the triazole C⁵-H appears to participate in a CH-O hydrogen bond with the carbonyl oxygen of Ile₁₂.¹² This observation is supported by the geometry and 2.2 Å distance between the atoms and is in agreement with the large dipole of the triazole ring.⁸ Hence, the normal i, i+4 hydrogen bonding that would have been provided to residues 12 and 18 in the parent pLI structure are replaced by the triazole ring. In addition, the amide NH of the ε-residue is hydrogen bonded to the carbonyl oxygen of Ile₁₂, while the N_δof the imidazole ring of His₁₇ is hydrogen bonded to the carbonyl of Ser₁₄. These structural features give rise to an increase in the α-helical pitch of about 1.8 Å in the region of the ε-residue. This increase in the local pitch creates a shallow pocket adjacent to the triazole that is occupied by a water molecule providing bridging hydrogen bonds that may contribute to the stability of the helical structure (Figure S5).

Figure 3.

(a) Detail from the crystal structure of 2 showing participation of the triazole residue in main chain hydrogen bonding; residues and nitrogen atoms of the triazole ring are numbered. (b) A top down view of the crystal structure of 3 showing the hydrophobic plate formed by the triazole ε² residues and inter-chain hydrogen bonds bridged by water. (c) Detail of two chains from the crystal structure of 3 showing the inter-helical crossing. Dashed lines indicate hydrogen bonds. In (a) and (c), H atoms are modeled based on N, C, and O coordinates.¹⁰

The ε²-amino acid substitution in peptide 3 creates an unusual right-handed interhelical crossover structure such that the helix formed by residues 1-22 of one chain is completed by residues 23-32 of another (Figure 3c, S6). The residues 20-24, which include the ε²-amino acid at position 22, act together as a template for the strand crossing, providing an overall helical chain register similar to that of the parent pLI fold. Inter-chain interactions consisting of a backbone hydrogen bond between the carbonyl oxygen of Glu₂₀ and the amide NH of Ala₂₃ and a water bridged hydrogen bond between N³ of the triazole ring and the amide NH of Arg₂₄ further stabilize this intertwined fold (Figure 3c). The isobutyl side chains of the ε²-residue also form a core hydrophobic packing in the helix bundle similar to the parent pLI (Figure 3b).

In summary, we have shown that a non-natural 1,2,3-triazole ε²-amino acid can replace a dipeptide in an α-helical secondary structure. In light of these observations, we suggest that Cu(I) catalyzed azide-alkyne coupling could be useful in the non-native chemical synthesis of peptides and proteins.¹²