Hierarchical cascades of instability govern the mechanics of coiled coils: helix unfolding precedes coil unzipping

Abstract

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.

Figure 1

(a) Schematic of the coiled coil used in the simulations consisting of three homo helical strands, denoted by P₁, P₂, and P₃, each having a sequence of Ac-EVEALEKKVAALESKVQALEKKVEALEHG-CONH₂. (b–d) Schematics of SMD simulations illustrating (b) pulling the Cα atoms of one end of the coiled coil while fixing the Cα atoms of the other end, and fixing all of the atoms on the two strands of the coiled coil while pulling the tip of the third strand in a direction (c) along the helix axis and (d) normal to the helix axis. The red and blue colors denote, respectively, the fixed and pulled segments of the coiled coil. To see this figure in color, go online.

Figure 2

Comparison of the SMD and MetaD simulation results for the free-energy landscape of unfolding of a single helix in the trimeric coiled coil. The SMD results were obtained by fixing the Cα atoms of one end of the coiled coil while pulling the Cα atoms of the other end of three helices and eventually partitioning the forces to the number of helices. The MetaD results were predicted along a reaction coordinate d₁, defined as the distance between the Cα atoms of the first and last residues of a single strand. To see this figure in color, go online.

Figure 3

(a–d) Results of SMD simulations showing the free-energy landscape of unzipping obtained by pulling the tip of a single strand (a and b) along the helix axis and (c and d) normal to the helix axis. The primary axes of plots a and c depict the energy, and the secondary axes illustrate the number of backbone hydrogen bonds (Hbonds) of the pulled strand. Panels b and d show snapshots of the coiled coil throughout the simulations. To see this figure in color, go online.

Figure 4

(a) Free-energy landscape of coil unzipping obtained by MetaD simulation of the coiled coil, with the CV d₂ defined as the distance between the backbone COM of one strand (say, P₁) from the backbone COM of the two other strands (say, P₂ and P₃). (b) The distance between the COMs of each heptad repeat of strand P₁ from the corresponding heptads of strands P₂ and P₃ as a function of simulation time. To see this figure in color, go online.

Figure 5

(a) Free-energy landscape of helix unfolding and unzipping obtained from the 2D MetaD simulation of the coiled coil with two CVs: d₁ (the distance between the Cα atoms of the first and last residues of one strand) and d₂ (the distance between the backbone COM of one strand from the COM of the two other strands). (b and c) Cross sections of the energy surface at (b) minimum d₁ and (c) minimum d₂, with a focus on a small deformation region (shown by the square box in a) around the energy dips. (d) Cross sections of the energy surface (along the dashed black and red lines illustrated in a) comparing the free energy required for partial unzipping of a single strand in a folded state versus a partially unfolded state. To see this figure in color, go online.