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. 2024 Oct 25;146(44):30252–30261. doi: 10.1021/jacs.4c09531

Coiled Coil Peptide Tiles (CCPTs): Expanding the Peptide Building Block Design with Multivalent Peptide Macrocycles

Anthony R Perez †, Adekunle Adewole †, Daphney Sihwa ‡, Michael E Colvin †, Andrea D Merg †,*
PMCID: PMC11544620  PMID: 39454098

Abstract

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Owing to their synthetic accessibility and protein-mimetic features, peptides represent an attractive biomolecular building block for the fabrication of artificial biomimetic materials with emergent properties and functions. Here, we expand the peptide building block design space through unveiling the design, synthesis, and characterization of novel, multivalent peptide macrocycles (96mers), termed coiled coil peptide tiles (CCPTs). CCPTs comprise multiple orthogonal coiled coil peptide domains that are separated by flexible linkers. The constraints, imposed by cyclization, confer CCPTs with the ability to direct programmable, multidirectional interactions between coiled coil-forming “edge” domains of CCPTs and their free peptide binding partners. These fully synthetic constructs are assembled using a convergent synthetic strategy via a combination of native chemical ligation and Sortase A-mediated cyclization. Circular dichroism (CD) studies reveal the increased helical stability associated with cyclization and subsequent coiled coil formation along the CCPT edges. Size-exclusion chromatography (SEC), analytical high-performance liquid chromatography (HPLC), and fluorescence quenching assays provide a comprehensive biophysical characterization of various assembled CCPT complexes and confirm the orthogonal colocalization between coiled coil domains within CCPTs and their designed on-target free peptide partners. Lastly, we employ molecular dynamics (MD) simulations, which provide molecular-level insights into experimental results, as a supporting method for understanding the structural dynamics of CCPTs and their complexes. MD analysis of the simulated CCPT architectures reveals the rigidification and expansion of CCPTs upon complexation, i.e., coiled coil formation with their designed binding partners, and provides insights for guiding the designs of future generations of CCPTs. The addition of CCPTs into the repertoire of coiled coil-based building blocks has the potential for expanding the coiled coil assembly landscape by unlocking new topologies having designable intermolecular interfaces.

Introduction

Molecular self-assembly is a defining feature of natural systems, wherein biomolecules organize into hierarchical structures via noncovalent interactions. The myriad of self-assembled architectures in nature showcases the incredible architectural diversity and function that can be achieved via bottom-up assembly methods and serves as inspiration for the development of synthetic biomaterials with emergent properties and functions that rival, or even surpass, their natural counterparts.1,2 Out of the library of biomolecules, proteins stand out as nature’s primary building block for assembling dynamic and functional architectures. Protein-based assemblies provide structural support to tissues and the extracellular matrix and carry out a variety of sophisticated functions (e.g., bacterial locomotion, molecular cargo transport, immune response, etc.)

While proteins serve as a logical starting point as molecular building blocks for the development of biomaterials,3 peptides represent an attractive synthetically accessible alternative.2 Despite their relatively short sequence length (typically <50 amino acids), peptides can replicate several features of proteins, including common protein folding motifs (e.g., α helices, β sheets, collagen triple helices, coiled coils, etc.) and various biological functions (e.g., cell-penetrating peptides, integrin binding, etc.).2,4−7 The truncated sequence length of peptides facilitates the establishment of sequence-to-structure relationships, which have been employed to construct an array of peptide-based architectures with diverse potential applications.7−9 Importantly, as fully synthetic constructs, peptides are amenable to the plethora of bioorganic chemical transformations that have been brought forward over the past several decades.10−13 The enhanced chemical diversity, afforded by the introduction of nonproteinogenic moieties, positions peptides as a highly modular biomolecular/biohybrid building block for constructing biomimetic materials with properties and functions that extend beyond the proteome.14,15

However, by virtue of their chain length, intrinsically, peptides cannot encode the same amount of structural and functional information that can be packaged into proteins, which comprise hundreds of amino acids. Peptides lack the ability to mimic complex tertiary structures, which define the protein’s three-dimensional (3D) topology and provide the intricate spatial framework of amino acids that drives their controlled assembly into multidimensional architectures. As a result, peptide-derived assemblies are often relegated to forming lower dimensional, and inherently less complex, structures (e.g., nanofibers, nanotubes, vesicles, etc.) via relatively nonspecific and nonprogrammable interactions. These limitations not only restrict the types of topologies that can be fabricated using short, linear peptides but also more importantly inhibit their rational assembly design and customization, which are required for developing peptide-based materials for targeted applications. Redesigning the peptide building block to overcome these limitations represents a potential step forward toward fully unlocking complex peptide-based architectures with tailorable structural features that better emulate the function and properties of natural protein-based assemblies.

So how can peptide building blocks be updated to increase their complexity and broaden their assembly scope and, thus, their application potential? One approach, which has not been well-explored, is to foray into the “gray area” of peptide/polypeptide design that lies between short, synthetically tractable peptides (<50 amino acids) and recombinantly expressed proteins (>150 amino acids; Figure 1a). Within this intermediate sequence design space, the complexity and function of peptide building blocks are markedly enhanced, as the longer sequence lengths afford the ability to encode the necessary amount of structural and functional information that is required for mimicking complex protein folds. And, importantly, these designs can still be accessed through chemical synthesis and are therefore amenable to the incorporation of nonproteinogenic moieties. Another approach is to continue exploring the assembly potential of cyclic peptides, in which the physical constraints of cyclization imbue multidirectional interactions through the outward display of chemically diverse amino acid side chains along the peptide circumference.16−19 Past work has primarily been limited to employing only short cyclic peptides (fewer than 12 amino acids) as assembly units.

Figure 1.

Figure 1

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(a) Sequence length of CCPTs lies between the lengths typically associated with traditional, short peptides and recombinantly expressed proteins. (b) CCPTs exhibit multidirectional interactions with the potential for building programmable, predetermined architectures (e.g., discrete 3D polyhedral architectures and extended nanostructures) via controlled associations along CCPT edges.

Here, we merge both approaches into a single peptide building block design, termed coiled coil peptide tiles (CCPTs; Figure 1b). CCPTs are protein-mimetic macrocycles (∼10 kDa; 96 amino acids) that comprise multiple equally spaced coiled coil-forming domains that are separated by flexible linkers. The constraints, imposed by cyclization, confer CCPTs with multivalent and multidirectional interactions (Figure 1b), in which each dimeric coiled coil-forming “edge” domain binds selectively with their designed, on-target peptide partner. Coiled coils, which consist of two or more α helices that intertwine to form a left-handed superhelical coil, were chosen as the assembly directing motif because of their well-established design rules that bestow the ability to engineer de novo sets of orthogonal dimeric coiled coils with excellent partner fidelity and tunable stability.20−34 These features have catalyzed the development of heterodimeric coiled coils as a modular and highly predictable intermolecular interaction for the fabrication of artificial bionanomaterials including polyhedral and spherical cages,35−38 and various geometrically defined nanostructures.32,39−44

We envision that this initial work will lay the foundation for employing CCPTs as a new versatile building block within the field of biomolecular self-assembly. CCPTs will serve as a welcome addition to the growing synthetic coiled coil assembly toolkit. Moreover, in the near-term, these structures have the potential to serve as multivalent, synthetic scaffolds for controlling the presentation of multiple different biologically relevant groups (e.g., protein–ligand receptors, antigens) with oligomeric and nanoscale control as shown previously with cyclic oligoproline trihelices.45

Results and Discussion

CCPT Design and Synthesis

Our initial efforts focused on the synthesis of “tile AAA” (TAAA), which consists of three coiled coil-forming peptide segments, A, that are separated by flexible nonhelical spacers (Figure 2a). The peptide sequence (A) originates from our previously published set of de novo orthogonal coiled coil peptide heterodimers.34 The orthogonal set comprises six 3.5-heptad peptides (A, B, C, A’, B’, and C’; Figure 2b) that form three orthogonal, parallel coiled coil heterodimers (AA’, BB’, and CC’; Figure 2c). The fidelity between coiled coil peptide partners is maintained as “on-target” pairings, i.e., AA’, BB’, and CC’, maximize the greatest number of favorable enthalpic contacts including interhelical hydrogen bonds between proximally positioned Asn residues (Asn residues are underlined in Figure 2b) and interhelical salt bridges between residues in the e and g positions. The vertices of TAAA are composed of two (Gly-Ser)3 linkers and a single GLPETG spacer, a vestige from employing enzyme-mediated cyclization (see below). The length chosen, (GS)3, stems from prior work on folded polypeptide nanotriangles, which determined that three Gly-Ser pairs is an optimum length that can fully accommodate the 120° angle.42

Figure 2.

Figure 2

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Peptide sequences. (a) Sequences of TAAA and TABC. (b) Sequences of de novo peptides that form orthogonal parallel coiled coil heterodimers. (c) A, A’, B, B’, C, and C’ form three on-target pairings: AA’, BB’, and CC’.

Initial attempts to synthesize TAAA on-resin using solid phase peptide synthesis (SPPS) were unsuccessful, as the length of the peptide extends beyond the practical limits of SPPS.46 Thus, we adopted a convergent synthetic strategy that employs native chemical ligation (NCL) to construct the linear peptide precursor (LAAA), which then undergoes Sortase A-mediated cyclization to yield TAAA (Figure 3a).

Figure 3.

Figure 3

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CCPT synthesis. (a) Convergent synthesis of TAAA (i: thioester formation, ii: NCL, iii: desulfurization, iv: SrtA-mediated cyclization). (b) Linear precursor, LAAA, is assembled from the NCL of F1 and F2, which are synthesized using SPPS. The synthesis and purification of TAAA were confirmed via (c) analytical high-performance liquid chromatography (HPLC) and (d) liquid chromatography mass spectrometry (LCMS).

NCL, which is widely utilized in the synthesis of polypeptides and proteins, involves reacting a N-terminal cysteinyl peptide with a C-terminal peptide thioester.47,48 Although no Cys are present in LAAA, we mutated a central Ala, which is omnipresent at b and c sites, with the foreknowledge that the incorporated Cys can be converted back to Ala via a desulfurization step.49 Two fragments, F1 and F2 (Figures 3b and S1 and Table S1), were synthesized using SPPS. F1 was synthesized on a Fmoc-hydrazine resin, which, under standard cleavage conditions, produces a hydrazide handle at the C-terminus.50 Activation of the hydrazide to the acyl azide in the presence of nitrous acid, followed by thiolysis with excess MPAA,51−53 yielded the peptide thioester (F1-MPAA; MPAA = mercaptophenylacetic acid), which was isolated by high-performance liquid chromatography (HPLC; Figure S2). NCL was performed between F1-MPAA and F2, and the embedded Cys was desulfurized to Ala to yield LAAA (Figure S3a–c and Table S1). The diglycine and LPETGG motifs, which are recognition sites for Sortase A (SrtA), at the C- and N-termini, respectively, were then ligated using SrtA-mediated cyclization to obtain TAAA.54−56 The monomeric cyclic construct (TAAA) represented the major product, which corroborates prior work that demonstrated the predilection for intramolecular cyclization over intermolecular oligomerization for long peptides (≥16 amino acids) under dilute conditions.54 Reverse phase HPLC and high-resolution liquid chromatography mass spectrometry (LCMS) confirmed the successful synthesis and isolation of TAAA (overall yield of 5%, see Supporting Information for details; Figure 3c,d).

Biophysical Characterization of TAAA, LAAA, and A

Circular dichroism (CD) spectroscopy was employed to elucidate differences among the free (A), linear (LAAA), and cyclized (TAAA) variants. Solutions of A (100 μM), LAAA (33 μM), and TAAA (33 μM) were prepared in PBS (pH 7.4). We note that all solutions were heated to 60 °C and cooled to 5 °C prior to conducting biophysical methods. CD spectra reveal a clear trend in which α helical secondary structure, assessed by the signal at 222 nm, increases in the order A < LAAA < TAAA (44, 71, and 85% helicity, respectively; Figure 4a and Table S2). We attribute this to the decreasing conformational freedom across the series, which minimizes the entropic cost associated with folding. Thermal denaturation studies, monitored via CD, confirm the increased helical stability of LAAA and TAAA with observed melting temperature (Tm) values of 57 and 56 °C, respectively, which is significantly higher than A (Tm = 18 °C; Figure 4b and Table S2).

Figure 4.

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Biophysical characterization of A', LAAA, TAAA, and their complexes. (a) CD spectra of A, LAAA, and TAAA. (b) CD thermal denaturation plots of A, LAAA, TAAA, AA’, L3AA’, and T3AA’. CD spectra of (c) A’, LAAA, and L3AA’ and (d) A’, TAAA, and T3AA’. (e) SEC traces of A, LAAA, and TAAA prior to the addition of A’ (bottom) and after the addition of A’ (top).

We next examined the coiled coil formation of LAAA and TAAA in the presence of A’ and compared these to free AA’. To solutions containing A, LAAA, and TAAA was added stoichiometric amounts of A’ (molar ratio of LAAA/A’ and TAAA/A’ = 1:3) in PBS (pH 7.4) to form AA’ and the proposed L3AA’ and T3AA’ complexes (Figure 4c,d). In each case, the concentration of the AA’ dimer (whether free or formed off LAAA and TAAA) was equivalent (100 μM). CD spectra of all three mixtures reveal an increase in helicity after the addition of A’ (Figure 4c,d and Table S2), which is consistent with the enhanced helical stability afforded by coiled coil formation. Moreover, an increase in the ratio of θ222/θ208 to values above 1 is observed for solutions that contained A and LAAA (Table S2). An increase in the θ222/θ208 value for solutions comprising α helices has been used as a proxy for coiled coil formation.57,58 In general, values close to 0.9 suggest the predominance of single chain helices, while values close to 1.0 suggest the predominance of coiled coils in solution.59−61 The similar θ222/θ208 values for TAAA and T3AA’ are attributed to intra- or intermolecular interactions between the helical domains of TAAA, prior to the addition of A’ (Table S2; see below).

CD thermal unfolding plots for all mixtures display a sigmoidal curve that is representative of a single cooperative unfolding event (Figure 4b). Surprisingly, the solutions containing L3AA’ and T3AA’ exhibit similar thermal stabilities (Tm of 51 and 48 °C, respectively) to free AA’ (Tm = 51 °C), which implies that the constraints enforced by cyclization does not significantly influence the coiled coil stability of AA’ within TAAA. The helicity between the different complexes follows the same trend observed for the CCPT constructs in the absence of A’ (68, 88, and 91% helicity for AA’, L3AA’, and T3AA’, respectively; Table S2). Forward and reverse melts on T3AA’ reveal identical thermal transition temperatures, which suggests the presence of a single thermodynamic minimum (Figure S4).

Size-exclusion chromatography (SEC) confirms that TAAA and A have the largest and smallest apparent sizes, respectively (Figure 4e). The more compact nature of LAAA, compared to TAAA, highlights the high degree of intramolecular self-association within the uncyclized biopolymer (Figure 4e, bottom). These nonspecific interactions likely contribute to the increased helical stability of LAAA as determined from CD. Furthermore, a shoulder peak, in addition to the main peak, is observed for TAAA, which suggests the presence of two different populations of TAAA that are similar in size. After the addition of A’, the relative size between LAAA and TAAA are exchanged, with the former eluting at lower elution volumes (Figure 4e, top). Unsurprisingly, we observe little change in elution volumes for AA’ and T3AA’, as coiled coil formation with A’ does not significantly alter the size of both complexes. The shift to a larger hydrodynamic size of LAAA upon complexation with A’ is attributed to the inhibition of nonspecific intramolecular interactions due to coiled coil formation. This, in turn, unravels LAAA and increases its physical hydrodynamic footprint (see the MD simulation results below). Judging by the elution volume for T3AA’, which closely aligns with the elution volume of the main peak of TAAA, we reason that TAAA exists as a dimer in solution with the shoulder peak (at slightly higher elution volumes) likely corresponding to the monomeric population of TAAA. Moreover, although L3AA’ and T3AA’ represent the major complexes assembled in solution, the minor peak at ∼15 mL is consistent with the elution volume for coiled coil dimers (e.g., AA’, BB’, CC’),34 which may suggest the presence of a small population of A’A’ (or free A’), at least under the more dilute conditions of SEC.

Biophysical Characterization of TABC

Encouraged by the successful assembly of T3AA’ we turned our attention toward building CCPTs that comprise edges that can be independently controlled, i.e, each CCPT edge contains a different orthogonal coiled coil peptide domain. We synthesized “tile ABC” (TABC; Figures 2a and 5a), which contains three different coiled coil peptide sequences (A, B, and C; Figure 2b). B and C orthogonally bind to B’ and C’ to form BB’ and CC’, respectively (Figure 2c).34 The synthesis of TABC was achieved using the same strategy employed for constructing TAAA with one small change: a one-pot ligation and desulfurization step was employed using imidazole, which does not interfere with the Cys desulfurization step (Figures S5–S7 and Table S1).62 The overall yield for TABC improved to 10% (see SI for details).

Figure 5.

Figure 5

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Characterization of TABC and TABC complexes. (a) LCMS spectrum of TABC. (b) CD spectra of TABC, A’, B’, C’, and TAA’BB’CC’. (c) CD thermal denaturation plots of TABC and TAA’BB’CC’. (d) Visual representation of the various dimeric and multimeric TABC complexes that were prepared. (e) SEC traces of TAA’BB’CC’, TABCC’, TABB’C, TAA’BC, and TABC. (f) Analytical HPLC traces of SEC fractions containing the major species in solution. Integration of the peaks confirm the expected stoichiometric ratios of TABC and free peptides (see Table S4).

Similar to TAAA, CD data (50 μM in PBS buffer, pH 7.4) confirm that TABC is helical (79% helicity), and after an equimolar addition of A’, B’, and C’, a slight increase in helicity (79 to 88%) and θ222/208 (1.00 to 1.02) are observed (Figure 5b and Table S3), which suggests coiled coil formation between A’, B’, and C’, and TABC. However, in contrast to TAAA, which comprises three identical coiled coil segments, CD thermal unfolding curves of TABC reveal multiple transitions owing to the presence of multiple different coiled coil-forming peptides (Figures 5c and S8). Although the first transition, centered at ∼15 °C, is commensurate with the observed Tm of A (17 °C) under identical conditions (50 μM), the second transition (∼59 °C) is markedly higher than Tm of B (<10 °C) or C (24 °C; Figures 5c and S8 and Table S3). The higher transition temperature likely stems from a combination of the increased stability associated with cyclization (similar to what we observed for TAAA) and nonspecific intra- or intermolecular interactions between coiled coil domains within CCPTs and between CCPTs in solution, respectively. The thermal denaturation plot of a solution containing equimolar concentrations of TABC, A’, B’, and C’ (50 μM each) also displays two visible thermal transitions at 33 and 70 °C (Figures 5c and S8 and Table S3). Unlike T3AA’, these transitions do not correlate with Tm values of individual solutions of AA’, BB’, and CC’ (Figure S9 and Table S3). The broad thermal denaturation profile suggests that the unfolding of AA’, BB’ and CC’ with TABC is to some extent independent of one another and does not unfold cooperatively. Moreover, there may be nonspecific interactions between the proximally placed coiled coil domains that could contribute to the higher thermal transition temperature.

To deconvolute the thermal melting profile of the presumed TAA’BB’CC’ complex, CD thermal denaturation experiments were carried out for solutions containing TABC with a single free peptide partner A’, B’, or C’ to form TAA’BC, TABB’C, and TABCC’, respectively. In all cases, we observe an increase in helicity and θ222/208 ratio for the bimolecular mixtures (Figures S10–S12 and Table S3). CD thermal denaturation curve of TAA’BC exhibits a single, broad transition centered at ∼45 °C, which is close to the observed Tm of AA’ (Tm = 51 °C) and T3AA’ (Tm = 48 °C; Figure S10b,c). However, for TABB’C and TABCC’, multiple, broad thermal transitions are observed with Tm values that do not align with BB’ and CC’ (Tm of 14 °C/70 °C and 27 °C/62 °C for TABB’C and TABCC’, respectively; Figures S11 and S12). The convoluted thermal melting curves contrast the melting curves obtained for T3AA’, in which a single thermal transition that was similar to free AA’, was observed. We surmise that the CD thermal denaturation profiles of the bimolecular complexes are a result of promiscuous interactions between the free peptides (A’, B’, or C’) and TABC, as well as intra- and intermolecular interactions of TABC, which can further stabilize the helical domains. This is in line with our prior work in which certain “off-target” pairs provide some additional stability.34

As a first step toward verifying the assembly of the proposed complexes, SEC was conducted on solutions containing TABC with and without the various free coiled coil peptide partners (Figure 5d). TABC elutes at a similar elution volume to TAAA and LABC (Figures 5e and S13), indicating that all three constructs have a similar hydrodynamic size. The relatively broad peak of TABC suggests that TABC may exist as monomers and dimers in solution, similar to the case for TAAA. The SEC trace of an equimolar mixture of TABC, A’, B’, and C’ in PBS (pH 7.4) reveals a major peak that elutes at a slightly lower elution volume than what was observed for TABC (Figure 5e). The slightly earlier elution volume and narrower peak width are consistent with the expected slight increase in the physical dimension and greater rigidity of TAA’BB’CC’ compared to TABC. As discussed above, the small peak at ∼15 mL suggests the minor presence of off-target coiled coil pairings, at least under SEC conditions. Analytical HPLC analysis of the fraction containing the major species in solution confirms the presence of TABC, A’, B’, and C’, with the expected 1:1:1:1 stoichiometry (Figure 5f and Table S4). In addition to analyzing the tetrameric complex (TAA’BB’CC’), SEC and analytical HPLC were also performed on binary solutions (TABC + A’/B’/C’; Figure 5d), and for each case, we observe major species in solution that comprise both components with equivalent molar ratios (Figure 5e,f and Table S4).

While SEC confirms the correct composition of TAA’BB’CC’, we conducted fluorescence quenching assays to verify that the free peptides (A’, B’, and C’) are dimerized to their designed coiled coil partner domain within TABC. We constructed three new CCPT variants in which 4-cyanophenylalanine (4CF) was installed at a single e position within the A (TA*BC), B (TAB*C), and C (TABC*) domains of TABC (Figures S14–S16 and Table S1). In addition, we synthesized corresponding peptides that incorporated selenomethionine (Mse) at a single a position for each complementary peptide (Mse-A’, Mse-B’, Mse-C’; Figure S17 and Table S1). 4CF, which fluoresces at 290 nm, is quenched when in close proximity to selenomethionine.63−65 As a result, fluorescence quenching can be used to probe the formation of parallel coiled coil heterodimers within TABC.

Separate solutions containing TA*BC, TAB*C, and TABC* were mixed with their on-target partners Mse-A’, Mse-B’, and Mse-C’ in PBS buffer (pH 7.4), respectively, and were heated and then cooled to 5 °C. As expected, fluorescence quenching at 290 nm (up to 48%) confirms the formation of on-target coiled coil heterodimers within the CCPTs (Figure S18). However, a significant amount of quenching is observed for control experiments in which the CCPT variants were mixed with off-target peptides. This is most notable for solutions containing Mse-C’, in which a 33% and a 36% decrease in fluorescence are observed when added to TA*BC and TAB*C, respectively, with the latter nearly matching the percent reduction in fluorescence that is observed for the on-target Mse-B’ (37%; Figure S18). These results verify the formation of promiscuous interactions and likely explain the convoluted CD thermal denaturation profiles for complexes containing TABC. We hypothesized that the presence of all free peptide partners (A’, B’, and C’) in the solution may restore the fidelity of the designed on-target heterodimers. Fluorescence quenching experiments were repeated, in which 4CF-incorporated CCPTs (TA*BC, TAB*C, TABC*) were mixed with both Mse- and non-Mse peptides. As shown in Figure 6, the fidelity between on-target peptide partners is restored with minimal quenching observed for control experiments (1–11%) and enhanced binding observed between the designed on-target partner (56–73%). These results indicate that partner fidelity is maintained when all of the peptide partners are present in the solution.

Figure 6.

Figure 6

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Fluorescence quenching assays. Fluorescence quenching data for complexes comprising (a) TA*BC, (b) TAB*C, and (c) TABC*. Yellow stars and gray pentagons represent the sites of 4CF and Mse, respectively, within the peptide sequences.

Molecular Dynamics (MD) Simulations of CCPT and CCPT Complexes

MD simulations were performed on TAAA, TABC, and the various CCPT complexes (T3AA’, TAA’BC, TABB’C, TABCC’, and TAA’BB’CC’). For each experiment, 400 ns simulations were carried out at 300 K in triplicate using the GROMACS software package and the AMBER99SB-ILDN force field.66,67 To obtain the starting structures of the free tiles, TAAA and TABC were simulated initially with bond restraints between the Cα positions on each helix domain before these were removed and simulated for 400 ns unrestrained. In contrast, the CCPT complexes were built by superimposing coiled coil dimers (AA’, BB’, CC’) from our previous study onto the prebuilt tile structure and were simulated unrestrained (Figures 7a,b, and S19).

Figure 7.

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MD simulation analysis of CCPTs and CCPT complexes. (a) Representative final MD simulation trajectories (400th ns) for all seven CCPTs and CCPT complexes. (b) Overlayed MD trajectories of 200–395 ns structures (Δt = 5 ns). (c) Calculated fraction of helical residues (out of the expected number of helical residues) within TAAA or TABC for all CCPTs and CCPT complexes studied. Average SASA for (d) TAAA (black line) and TAAA within T3AA’ (red line) and (e) TABC (black line) and TABC within TAA’BB’CC’ (blue line).

All simulations show good convergence after 200 ns based on root-mean-square deviation (RMSD) analysis (Figures S20 and S21). The large RMSD values (>1 nm) for all seven structures are a result of the significant deviation of the final simulated trajectory compared to the initial, idealized structures that were built in Pymol. Root-mean-square fluctuations (RMSF) of TAAA and TABC highlight the increased mobility of the linker regions compared to the helical coiled coil-forming domains, although notable mobility was still observed for the latter (Figure S22a,c). The helical domains shift to lower RMSF values upon the introduction of their complementary peptides, which is a result of the added stabilization associated with coiled coil folding (Figure S22b,d–g).

To correlate the reduction in RMSF with an increase in the α-helical content, we compared the number of α-helical residues in the entire structure (from the MD simulations) with the total number of expected helical residues (i.e., the total number of residues minus the linker regions; see the SI for details) to yield the helical fraction for each CCPT and CCPT complex (Figures S23 and S24). Analysis of the helical fractions for each construct reveals an increase in percent helical content for T3AA’ and TAA’BB’CC’ compared to TAAA and TABC, respectively (Figure S25). Interestingly, the bimolecular complexes exhibit a fraction of helical residues similar to those of T3AA’ and TAA’BB’CC’ (Figure S25), which may be a result of the decreased entropic penalty for folding due to the straightening of a single tile edge. With the exception of TAAA and TABC, the helical fractions from MD simulations correspond closely to the % helical content that was derived from CD (Tables S2 and S3) and recapitulates the same general trends (Figure S25c). The higher percentage helicity calculated from CD for TAAA and TABC provides further evidence that free CCPTs nonspecifically interact in situ, which agrees with the results obtained from SEC that indicate that CCPTs are likely present as dimers in solution. Such face-to-face dimerization may explain the discrepancy in the helical stability of the coiled coil peptide domains between the CD and MD simulation data. In addition, we note that more simulations may need to be conducted to confirm these differences. The difference in helicity between CCPTs (TAAA and TABC) and the complexed tiles are more apparent when analyzing just the changes in the helical fraction of TAAA and TABC. A 52% and a 46% increase in the helical fraction for TAAA and TABC are observed after complete CCPT complexation, which highlights the increased secondary structure of the coiled coil-forming domains of CCPTs upon coiled coil formation with their designed on-target peptide partner (Figures 7c, S26, and S27).

From our previous work, MD simulations revealed a decrease in the number of interhelical contacts (hydrogen bonds and salt bridges) for BB’ compared to AA’ and CC’, which provided a rationale for the substantially lower thermal stability of the former heterodimer.34 Interested in identifying any differences at the coiled coil interface that may arise due to cyclization, we analyzed the interfacial contacts (AA’, BB’, and CC’) of the heterodimers formed on TAAA and TABC for both the bimolecular and tetrameric complexes (Figures S28–S30). MD results reveal slight differences with modest 6 and 18% absolute changes, on average, for the average number of hydrogen bonds and salt bridges, respectively. However, these changes are not statistically significant. Furthermore, there was no trend observed, with approximately half of the dimeric coiled coil interfaces showing an increase in interhelical contacts and the other half showing a decrease in interhelical contacts, which suggests that there is no apparent difference between the cyclized and uncyclized heterodimers.

Observations of the trajectories for TAAA and TABC uncover the preference for forming “T-shape” geometries in which two of the helical domains make significant contacts along their helix at the expense of the third (Figures 7a,b and S19). MD simulations of TABC disclose preferential interactions between the B and C domains. The significant degree of nonspecific intramolecular interactions can explain the broad CD thermal denaturation curves that we observed for TABC (Figure 5c). In contrast to the collapsed structures of TAAA and TABC, MD trajectories of T3AA’ and TAA’BB’CC’ more closely resemble the “open-core” triangular constructs that served as the initial structures (Figures 7a,b and S19). The expanded structures of T3AA’ and TAA’BB’CC’ are confirmed from radius of gyration calculations, which show a 37% and a 15% expansion, respectively, in comparison to their free counterparts (Figure S31). Increased radius of gyration is also observed for the bimolecular complexes, as the presence of A’, B’, or C’ eliminates the intramolecular B/C interactions that lead to the collapsed structure (Figures 7a,b, S19, and S31). These results underline the reduced propensity for nonspecific intramolecular interactions when free peptide partners are bound to their on-target binding domain and highlight the increased rigidity of the complex associated with coiled coil folding along the tile edges. We further note that additional MD experiments, carried out for LAAA and L3AA’, corroborate the significant size change that was observed from SEC after the addition of 3 equivalence of A’ to LAAA (Figures 4e and S32).

The contraction of TAAA and TABC in the absence of their free binding partners was further verified through analysis of the solvent-accessible surface area (SASA; Figures 7d,e and S33). The free tiles (TAAA and TABC) have the lowest SASA among the simulated structures. On the other hand, the SASA for TAAA and TABC within the CCPT complexes is markedly more solvent-exposed, which is notable considering that the coiled coil domains are bound to free peptides along the coiled coil interface. This correlates well with the restoration of the open triangular structure that is formed as a result of the rigidification inherent in coiled coil folding. We observed intermediate SASA values for the three bimolecular systems (TAA’BC, TABB’C, and TABCC’) that were between TABC and TAA’BB’CC’, which indicates that the presence of a single complementary coiled coil partner partially restores the triangular geometry (Figure S33).

Conclusions

We establish the design, synthesis, and characterization of a new, multivalent peptide construct, termed coiled coil peptide tiles (CCPTs). CCPTs were constructed via a fragment coupling approach using NCL and enzyme-mediated cyclization. To the best of our knowledge, the synthesis of TAAA and TABC represents the largest cyclic peptides that have been synthesized via chemoenzymatic methods. We demonstrate that CCPTs possess multidirectional facing edges that can be selectively engaged through orthogonal coiled coil formation with each edge domain’s free coiled coil peptide binding partner. A suite of biophysical techniques confirm that the edges are amenable to coiled coil formation and that cyclization does not meaningfully perturb the coiled coil fold along the tile edges. Importantly, cyclization does not interfere with the fidelity between designed on-target peptide partners if all free binding partners are present in the mixture. MD simulation analysis confirms the experimental results, revealing little deviation between the cyclized and free systems in terms of the number of interfacial contacts present within the coiled coil heterodimers. Furthermore, MD simulation results reveal the rigidification and expansion of CCPTs upon complexation, i.e., coiled coil formation, with their designed binding partners. These trajectories were verified by increased SASA and radius of gyration values for the CCPT complexes in comparison to those of free CCPTs.

The presented work represents an advancement that pushes the bounds of synthetic peptide-based designs. To date, the vast majority of peptides that are employed for hierarchical self-assembly consist of short, linear peptides that lack the multivalency exhibited by proteins.7 We envision that CCPTs can serve as synthetically tractable constructs that mimic natural tertiary structural motifs (e.g., helix-turn-helix), which give rise to the 3D structure of globular proteins. From this perspective, these multidimensional constructs have the potential to be used for the design and assembly of fully synthetic protein-mimetic architectures having topologies outside the bounds of what can be assembled by using conventional linear peptides. Lastly, the ability to construct these biomacromolecules synthetically leaves the door open for the introduction of chemical moieties with properties and functions beyond what is found in the proteome. Current efforts are focused on assembling CCPTs into hierarchical assembly architectures. We foresee that CCPTs will find immediate application as a novel, multivalent building block for expanding the coiled coil assembly toolkit.

Acknowledgments

This work was supported by the NSF (CHE-2316870, A.D.M.), the ARO (W911NF-23-1-0365, A.D.M.), and the University of California, Merced. A.R.P. acknowledges fellowship support from the NSF-CREST: Center for Cellular and Biomolecular Machines at UC Merced (NSF-HRD-1547848 and NSF-HRD-2112675). We acknowledge computing time on the Pinnacles cluster at UC Merced (NSF-MRI-2019144). We thank Prof. Shahar Sukenik for helpful feedback on the fluorescence experiments. We also thank Joseph McTiernan for help with analyzing the MD results. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation herein.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c09531.

  • Detailed experimental procedures, synthesis protocols, MALDI-TOF MS, HPLC, CD, SEC, fluorescence, and MD simulation protocols and data (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ja4c09531_si_001.pdf (16.8MB, pdf)

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