Structural Determinants of Coiled Coil Mechanics

: The natural abundance of coiled coil (CC) motifs in the cytoskeleton and the extracellular matrix suggests that CCs play a crucial role in the bidirectional mechanobiochemical signaling between cells and the matrix. Their functional importance and structural simplicity has allowed the development of numerous applications, such as protein-origami structures, drug delivery systems and biomaterials. With the goal of establishing CCs as nanomechanical building blocks, we investigated the importance of helix propensity and hydrophobic core packing on the mechanical stability of 4-heptad CC heterodimers. Using single-molecule force spectroscopy, we show that both parameters determine the force-induced dissociation in shear loading geometry; however, with different effects on the energy landscape. Decreasing the helix propensity lowers the transition barrier height, leading to a concomitant decrease in the distance to the transition state. In contrast, a less tightly packed hydrophobic core increases the distance to the transition state. We propose that this sequence-structure-mechanics relationship is evolutionarily optimized in natural CCs and can be used for tuning their mechanical properties in applications.


ABSTRACT:
The natural abundance of coiled coil (CC) motifs in the cytoskeleton and the extracellular matrix suggests that CCs play a crucial role in the bidirectional mechanobiochemical signaling between cells and the matrix.Their functional importance and structural simplicity has allowed the development of numerous applications, such as protein-origami structures, drug delivery systems and biomaterials.With the goal of establishing CCs as nanomechanical building blocks, we investigated the importance of helix propensity and hydrophobic core packing on the mechanical stability of 4-heptad CC heterodimers.Using single-molecule force spectroscopy, we show that both parameters determine the forceinduced dissociation in shear loading geometry; however, with different effects on the energy landscape.Decreasing the helix propensity lowers the transition barrier height, leading to a concomitant decrease in the distance to the transition state.In contrast, a less tightly packed hydrophobic core increases the distance to the transition state.We propose that this sequence-structure-mechanics relationship is evolutionarily optimized in natural CCs and can be used for tuning their mechanical properties in applications.

3
Coiled coils (CCs) are self-assembled, superhelical motifs that are naturally found in numerous proteins in the cytoskeleton and the extracellular matrix. 1 CCs consist of two (or more) α-helices, each characterized by a repetitive pattern of seven amino acids, called heptad (abcdefg) n (Figure 1A).The helix propensity of the CC-forming peptides was calculated using AGADIR. 16ilizing this simple design, CCs serve as model systems for studying protein folding and stability.5] Considering their natural role as a mechanical scaffold, surprisingly little information is available about the sequence-structure-mechanics relationship of CCs.With the goal of introducing CCs as nanomechanical building blocks, we have characterized three different CC heterodimers with atomic force microscope (AFM)-based single-molecule force spectroscopy (SMFS; Figure 1B).We show that hydrophobic core packing and helix propensity affect the thermodynamic stability in similar ways; however, the underlying changes to the energy landscape are different.
was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted January 26, 2019.; https://doi.org/10.1101/530873doi: bioRxiv preprint Using the thermodynamically and mechanically well characterized CC A 4 B 4 as the starting point (Figure 1C), 5,17 we used the standard rules of CC design [2][3] to obtain one sequence with a reduced helix propensity and a second sequence with a different hydrophobic core packing.To reduce the helix propensity, Ala in the b position was substituted with Ser in all heptads 18 (A 4S B 4S ; Figure 1C).Hydrophobic core packing was altered using another β-branched amino acid in position a (Val instead of Ile; A 4V B 4V ).0] To define the points of force application, Cys was introduced at the desired termini for coupling the CC to the surface and the AFM cantilever.For A-peptides, Cys was located at the N-terminus, while it was placed at the C-terminus of B-peptides, thus establishing a shear pulling geometry (Figure 1B).were significantly destabilized (Table 1).The corresponding free energy difference between the folded (F) and the unfolded (U) state (ΔG F-U ) was obtained from van't Hoff plots (Figure S2). To address the question whether the thermodynamic and mechanical stabilities are correlated and how the respective substitutions affect the energy landscape, SMFS was carried out.The B-peptide was immobilized to the cantilever, while the corresponding A-peptide was immobilized to the surface (Figure S3).The CC only forms when the cantilever is in contact with the surface.When retracting the cantilever, the CC experiences a steadily increasing force until it ruptures (Figure 1B).At a retract speed of 400 nm s -1 , both A 4S B 4S and A 4V B 4V are mechanically less stable than A 4 B 4 (Figure 2A).At this retract speed, the most probable rupture forces decrease 20 pN and 15 pN for A 4S B 4S and A 4V B 4V , respectively.This provides a first indication that the substitutions also lower the mechanical stability of the CCs.Subsequent dynamic SMFS, performed over a range of loading rates (r = dF/dt) 23 from approximately 15 pN s -1 to 11000 pN s -1 , revealed that both modified CCs possess a lower mechanical was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted January 26, 2019.; https://doi.org/10.1101/530873doi: bioRxiv preprint stability over the complete range of loading rates (Figures S4-S6); however, their dependence on the loading rate is different.Fitting the data to the Bell-Evans model 23 (Figure 2B) allows for obtaining more detailed information about the energy landscape of the CC two-state system (folded CC vs. random coil peptides): k off , the force-free dissociation rate, and Δx F-TS , the distance from the folded to the transition state (TS).Using the Arrhenius equation, also the barrier height between the folded and the transition state (ΔG F-TS ) can be calculated, provided that the Arrhenius pre-factor is known.Here, we use an Arrhenius pre-factor of 5 ´ 10 8 s -1 , which was estimated for the dimeric GCN4 leucine zipper. 24Table 1 summarizes the obtained fit values, as well as the calculated ΔG F-TS values.Comparing the k off values of A 4S B 4S and A 4V B 4V to A 4 B 4 shows that both modifications lower the height of the transition state barrier (higher k off and lower ΔG F-TS ).This lower barrier height is was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted January 26, 2019.; https://doi.org/10.1101/530873doi: bioRxiv preprint 6 correlated with the different thermodynamic stabilities (Table 1) and helix propensities of the three CCs (Figure 1C).This suggests that a reduced helix propensity lowers the barrier height, thereby affecting both the thermodynamic and mechanical stability of the CCs.Interestingly, the Δx F-TS values do not correlate with the thermodynamic stabilities.The Ala-Ser modification (A 4S B 4S ) reduces Δx F-TS , whereas the Ile-Val modification (A 4V B 4V ) increases Δx F-TS .This suggests that modifications in the solvent-exposed residues affect the energy landscape of the CC interaction differently when compared to hydrophobic core modifications.6][27] Initially, the force increases linearly with extension and the helices remain intact (phase I).At a strain of 10-20 %, the individual helices start uncoiling at an almost constant force (phase II).In long CCs, the force increases sharply after the helices are uncoiled and the resulting structure is extended further (phase III).For CCs with a length of ≤4 heptads loaded in the shear geometry, the CC chains separate in phase I or just at the transition to phase II. 17This is a direct result of the chain separation mechanism.At loading rates typically used in SMFS, the applied force causes the uncoiling of helical structure at the points of force application.This, in turn, destabilizes the CC thermodynamically and facilitates the subsequent dissociation of the CC chains (uncoiling-assisted dissociation).This mechanism allows for explaining the effects of helix propensity and hydrophobic core packing on CC shearing.
was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted January 26, 2019.; https://doi.org/10.1101/530873doi: bioRxiv preprint When mechanically loaded in shear geometry, the hydrogen bonds stabilizing the individual helices are aligned parallel to the force vector, whereas the hydrophobic side chains are arranged almost perpendicularly.The torsional angles and helical propensities of the individual amino acids, which are responsible for maintaining stable hydrogen bonds in the helices, 18,28-29 are thus critically determining the resistance of CCs to shear forces.For CCs with a lower overall helix propensity less force is required to uncoil the individual helices.In addition, a lower helix propensity is correlated with a lower thermodynamic stability.Uncoiling of only small parts of helical structure thus destabilizes an already less stable CC further, an affect that was observed earlier when decreasing CC length. 17Assuming that the hydrophobic core is not altered, this suggests that chain separation occurs at smaller extensions.In the case of A 4S B 4S , a higher k off value is thus accompanied by a shorter Δx F-TS .This result is in line with the observation that artificial constraints, which stabilize the helices against uncoiling, lead to an increase in the forces required for chain separation. 10,30ollowing this line of argumentation, Δx F-TS for A 4V B 4V is expected to lie in between the values obtained for A 4S B 4S and A 4 B 4 ; however, A 4V B 4V shows an increase in Δx F-TS .This suggests that substituting Ile with Val does not only affect the helix propensity.It also causes a less tightly packed and more flexible CC interface.The increased Δx F-TS can thus be explained as follows: the pre-existing flexibility at the hydrophobic core permits a more dynamic rearrangement of the Val side chains in was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.

2 Figure 1 .
Figure 1.Experimental design.A) CC heptad pattern.B) SMFS setup showing mechanical loading of a CC heterodimer in the shear geometry.C) Sequences of the CCs used in this study.The terminal cysteines define the shear pulling geometry.The helix propensity of the CC-forming peptides was calculated using AGADIR.16 Figure S1.As expected, A 4 B 4 shows the highest melting temperature T m , while A 4S B 4S and A 4V B 4V

Figure 2 .
Figure 2. Single-molecule force spectroscopy.A) Representative rupture force histograms obtained at a retract speed of 400 nm s -1 , with n = 284 (A 4 B 4 ), n = 243 (A 4S B 4S ) and n = 420 (A 4V B 4V ).The dashed lines show Gaussian fits, applied to extract the most probable rupture forces.The inset shows representative force-distance curves for each CC.B) Dynamic SMFS plot.Each CC was measured in triplicate using a different cantilever and surface (different shades of the same color).The solid lines represent fits to the Bell-Evans model.

Figure 3 .
Figure 3. Energy landscape of the CCs.The horizontal line represents the distance from the folded (F) to the transition state (TS) (Δx F-TS ), while the vertical solid arrow represents the transition barrier height (ΔG F-TS ).The dotted arrow shows the energy difference between the folded and the unfolded state (ΔG F-U ).
• Mechanical properties • Rational design • Single-molecule studieswas not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.