The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.     

Multiple glycosylation of de novo designed α-helical coiled coil peptides

The aim of this study was to investigate the influence of multiple O-glycosylation in α-helical coiled coil peptides on the folding and stability. For this purpose we systematically incorporated one to six β-galactose residues into the solvent exposed positions of a 26 amino acid long coiled coil helix. Surprisingly, circular dichroism spectroscopy showed no unfolding of the coiled coil structure for all glycopeptides. Thermally induced denaturations reveal a successive but relative low destabilization of the coiled coil structure upon introduction of β-galactose residues. These first results indicate that O-glycosylation of the glycosylated variants is easily tolerated by this structural motif and pave the way for further functional studies.     

Local destabilization of the tropomyosin coiled coil gives the molecular flexibility required for actin binding

Tropomyosin, a coiled coil protein that binds along the length of actin filaments, contains 40 uninterrupted heptapeptide repeats characteristic of coiled coils. Yet, it is flexible. Regions of tropomyosin that may be important for binding to the filament and for interacting with troponin deviate from canonical coiled coil structure in subtle ways, altering the local conformation or energetics without interrupting the coiled coil. In a region rich in interface alanines (an Ala cluster), the chains pack closer than in canonical coiled coils, and are staggered, resulting in a bend [Brown et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8496-8501]. Brown et al. suggested that bends at alanine clusters allow tropomyosin to wind on the actin filament helix. Another explanation is that local destabilization of the coiled coil, rather than close packing of the chains at Ala clusters per se, allows flexibility. Changing three Ala residues to canonical interface residues, A74L-A78V-A81L, greatly stabilized tropomyosin, measured using circular dichroism and differential scanning calorimetry, and reduced actin affinity >10-fold. Normal actin affinity and stability were restored in a mutant A74Q-A78N-A81Q that mimicked the stability of the Ala cluster but not the close packing of the chains. Analysis and modeling of comparable mutations introduced closer to the N-terminus show that the effects on stability and function depend on context. Models based on tropomyosin crystal structures give insight into possible effects of the mutations on the structure. We conclude that the significance of the Ala clusters in allowing flexibility of tropomyosin is stability-driven.     

Alpha-helix to random-coil transitions of two-chain coiled coils: the use of physical models in treating thermal denaturation equilibria of isolated subsequences of alpha alpha-tropomyosin

Two extant models of thermal folding/unfolding equilibria in two-chain, alpha-helical coiled coils are tested by comparison with experimental results on excised, isolated subsequences of rabbit alpha alpha-tropomyosin (Tm). These substances are designated iTmj where i and j are, respectively, the residue numbers (in the 284-residue parent chain) of the N- and C-terminal residues of the subsequence. One model postulates that a coiled coil consists of segments, each denaturing in an all-or-none manner, like small globular proteins. Thus this model yields a small number of populated molecular species. In an extant calorimetry study of 11Tm127 and of 190Tm284, each required only two all-or-none-segments, and their enthalpies and transition temperatures were assigned. These assignments are shown here to yield the concentration of all molecular species, and therefore the helix content, as a function of temperature. Such calculations for 190Tm284 are in tolerable agreement with CD experiments, but those for 11Tm127 are in gross disagreement. Thus, either the model itself or the calorimetric assignment is faculty. In the second model, all conformational states are counted and weighted, as in the Zimm-Bragg theory for single-chain polypeptides. This theory has been extended (by Skolnick) to two-chain coiled coils and is here used to fit CD data for 11Tm127, 142Tm281, and 190Tm284. The fit is tolerable for 11Tm127, good for 142Tm281, and quantitative for 190Tm284. Thus this comparison does not falsify this second model. The helix-helix interaction free energy, obtainable from the fit, shows nonadditivity when isolated subsequences are compared with the parent. This suggests that removal of a region from a long coiled coil allows energetically substantial adjustments in side-chain packing in the helix-helix interface. Thus, the helix-helix interaction in long coiled coils is characteristic of a global free energy minimum and not just of the regional constellation of side chains.     

Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils

Membrane-embedded protein domains frequently exist as α-helical bundles, as exemplified by photosynthetic reaction centers, bacteriorhodopsin, and cytochrome C oxidase. The sidechain packing between their transmembrane helices was investigated by a nearest-neighbor analysis which identified sets of interfacial residues for each analyzed helix–helix interface. For the left-handed helix–helix pairs, the interfacial residues almost exclusively occupy positions a, d, e, or g within a heptad motif (abcdefg) which is repeated two to three times for each interacting helical surface. The connectivity between the interfacial residues of adjacent helices conforms to the knobs-into-holes type of sidechain packing known from soluble coiled coils. These results demonstrate on a quantitative basis that the geometry of sidechain packing is similar for left-handed helix–helix pairs embedded in membranes and coiled coils of soluble proteins. The transmembrane helix–helix interfaces studied are somewhat less compact and regular as compared to soluble coiled coils and tolerate all hydrophobic amino acid types to similar degrees. The results are discussed with respect to previous experimental findings which demonstrate that specific interactions between transmembrane helices are important for membrane protein folding and/or oligomerization. Proteins 31:150–159, 1998. © 1998 Wiley-Liss, Inc.     

Application of the augmented theory of alpha-helix-to-random-coil transitions of two-chain, coiled coils to extant data on synthetic, tropomyosin-analog peptides

The statistical mechanical theory for the helix-to-random-coil transition in two-chain coiled coils is applied to extant data for two synthetic coiled-coil polypeptides. These peptides have the primary structure K(LEALEGK)_n, in which n = 4, 5. This repeating heptet sequence mimics the pattern of hydrophobic, acidic, and basic residues characteristic of the 284-residue tropomyosin molecule, the prototypical coiled-coil protein. Theoretical calculations for single chains show that such model peptides cannot be directly compared to proteins like tropomyosin because of differences in chain length (29 and 36 residues vs 284) and in intrachain interactions, the latter caused by the differences in amino acid composition and seqeunce between protein and model. Application of the theory to extant data on the two synthetic peptides provides a semiquantitative fit and results in an assessment of the interhelix interaction in the model peptides. The value obtained, ～ 2000 cal · (mol of turn pairs)^–1, is four to five times larger than has been obtained for tropomyosin. This probably is a result of greater regularity in the structure of the synthetics and of the exclusive presence of leucine in the hydrophobic interface. The theory employed here insists that this powerful interhelix interaction in the synthetic is the principal reason that such short chains can be so highly helical at moderate and low temperatures. Theory predicts, indeed, that a tropomyosin-length chain with a sequence homologous to these synthetics would be completely thermally stable in the entire temperature range accessible in aqueous solutions. Theory also predicts a much more pronounced effect of concentration on the 29- and 36-residue synthetic polymers than is predicted or observed in the case of tropomyosin, and it also predicts a pronounced stabilizing effect of pH-reduction on the thermal curves. On the last two points, sufficient data are not yet available with which to test the theory.     

CD and (13)C(alpha)-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of alpha-tropomyosin

Synthesis and CD and (13)C(alpha)-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide ((190)Tm(254)) comprising residues 190-254 of the alpha-tropomyosin chain. CD on a version disulfide cross-linked via the N-terminal cysteine side chains indicates that this dimer is highly helical and melts near 48 degrees C. The CD is independent of peptide concentration, showing that association of (190)Tm(254) stops at the two-strand level. Similar studies on the reduced version show much lower helix content at low temperature, melting points below room temperature, and the expected concentration dependence. The observed melting temperature of the reduced peptide is far below (by 27 degrees C) that expected from an extant analysis of calorimetry data on parent tropomyosin that designates (190)Tm(254) as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for (190)Tm(254) are combined with extant data for other subsequences argue decisively against the concept of specific independently melting blocks within the tropomyosin chain. The data for (190)Tm(254) also serve to test recent ideas on the sequence determinants of structure and stability in coiled coils. Analysis shows that some ideas, such as the stabilizing effect of leucine in the d heptad position, find support, but others--such as the destabilizing effect of alanine in d, the dimer-disfavoring effect of beta-branching in d and its dimer-favoring effect in a, and the dimer-directing effect of asparagine in a--are more questionable in tropomyosin than in the leucine zipper coiled coils. (13)C(alpha)-NMR data at two labeled sites, L228(d) and V246(a), of (190)Tm(254) display well-separated resonances for folded and unfolded forms at each site, indicating that the transition is slow on the NMR time scale and thus demonstrating the possibility of obtaining thermodynamic and kinetic information on the transition at the residue level.     

α-Helical assembly of biologically active peptides and designed helix bundle protein

The formation of α-helical assembly by complexing biologically active peptides with de novo designed protein is described. The de novo designed protein described here is a cystinelinked 4-helix bundle protein constructed with 80 amino acid residues and forms a hydrophobic core region surrounded by 4 helices in an aqueous solution. The biologically active peptides, such as melittin and human growth hormone releasing factor, contain the sequences that are able to form amphiphilic helices. These peptides alone do not form the α-helix structure in a diluted solution with low ion strength. But on mixing with the designed helix bundle protein, the peptides are strongly bound to the protein with the induction of α-helical structure in the biologically active peptides. The content of induced α-helix is in accord with that estimated from the amphiphilic sequence. The results mean that a novel architecture composed of α-helices is formed. Fluorescent and temperature-scanning measurement revealed that the α-helical assembly is constructed with hydrophobic interaction. Also, it is shown by means of fluorescence depolarization that the assembly has a compact globular form corresponding to 1 : 1 complex. © 1994 John Wiley & Sons, Inc.     

Probing the influence of sequence-dependent interactions upon α-helix stability in alanine-based linear peptides

The observation that short, linear alanine-based polypeptides form stable α-helices in aqueous solution has allowed the development of well-defined experimental systems with which to study the influence of amino acid sequence upon the stability of secondary structure. We have performed detailed conformational searches upon six alanine-based peptides in order to rationalize the observed variation in the α-helical stability in terms of side-chain-backbone and side-chain-side-chain interactions. Although a simple, gas-phase, potential model was used to obtain the conformational energies for these peptides, good agreement was obtained with experiment regarding their relative α-helical stabilities. Our calculations clearly indicate that valine, isoleucine, and phenylalanine residues should destabilize the α-helical conformation when included within alanine-based peptides because of energetically unfavorable side-chain-backbone interactions, which tend to result in the formation of regions of 3₁₀-helix. In the case of valine, the destabilization most probably arises from entropic effects as the isopropyl side chain can assume more orientations in the 3₁₀-helical form of the peptide. A detailed examination of very short-range interactions in these peptides has also indicated that an interaction, involving fewer than five consecutive residues, whose stabilizing effect reinforces that of the (i, i + 4) hydrogen bond may be the basis of the requirement for increased nucleation (σ) and propagation parameters (s) required by Zimm–Bragg theory to predict the α-helical content for compounds in this class of short peptides. Our calculations complement recent work using modified Zimm–Bragg and Lifson–Roig theories of the helix–coil transition, and are consistent with molecular dynamics simulations upon linear peptides in aqueous solution. © 1993 John Wiley & Sons, Inc.     

The double helix coiled coil structure of murein lipoprotein from Escherichia coli.

A rod-like structure is proposed for the murein lipoprotein of Escherichia coli, built of two parallel unbroken α-helices arranged in a coiled coil of the same type as in the muscle protein tropomyosin. The amino acid sequence has the required regular pattern of hydrophobic amino acids at intervals of three and four residues and the secondary structure predicted from the sequence is 80% helical. A space-filling model confirms that the coiled coil model is stereochemically reasonable, and energy calculations for a series of coils with different radii suggest that the best structure is one with the helix axes 8.25 Å apart. Energyrefined atomic co-ordinates have been calculated which show that the hydrophobic side-chains form a series of close-packed unstrained contacts between the two helices along the entire length of the sequence. On the basis of this study the hexagonal membrane pore model and the segmented helix model proposed by others seem unlikely. The coiled coil has a strongly hydrophilic outer surface, suggesting that the protein has a watery environment within the E. coli cell envelope and is not strictly a membrane protein. Probably only the fatty acid portion of the lipoprotein penetrates into the lipid region of the outer membrane, so that the protein may act as a tie or a spacer between the lipid and the murein wall.     

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ?-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.     

Some properties of acid-reassembled tropomyosin

The preference for parallelism of the two chains in tropomyosin coiled coils is thought to result from interchain salt bridges. To examine this idea, studies are presented of tropomyosin molecules reassembled from chaotropic solvents in acid solution, where cross-links cannot exist. The acid-reassembled molecules are appreciably less disulfide cross-linkable in acid than native molecules, a result explainable if some antiparallel dimers indeed form at low pH. Physical studies (backbone- and tyrosine-region CD and intrinsic viscosity) indicate that refolding in acid yields a molecular population demonstrably different in tyrosine-region CD from native, but having comparable (but not identical) helix content, thermal stability, and dimensions. Moreover, the refolding in acid after either thermal or chaotropic-solvent denaturation yields the same final state, arguing that it is an equilibrium state. All these results are consistent with, but do not prove, that the acid-reassembled population includes an appreciable fraction (2/3) of antiparallel coiled-coil dimers. © 1995 John Wiley & Sons, Inc.     

Crystal structure of the amino-terminal coiled-coil domain of the APC tumor suppressor

Coiled coils serve as dimerization domains for a wide variety of proteins, including the medically important oligomeric tumor suppressor protein, APC. Mutations in the APC gene are associated with an inherited susceptibility to colon cancer and with approximately 75 % of sporadic colorectal tumors. To define the basis for APC pairing and to explore the anatomy of dimeric coiled coils, we determined the 2.4 A resolution X-ray crystal structure of the N-terminal dimerization domain of APC. The peptide APC-55, encompassing the heptad repeats in APC residues 2-55, primarily forms an alpha-helical, coiled-coil dimer with newly observed core packing features. Correlated asymmetric packing of four core residues in distinct, standard rotamers is associated with a small shift in the helix register. At the C terminus, the helices splay apart and interact with a symmetry-related dimer in the crystal to form a short, anti-parallel, four-helix bundle. N-terminal fraying and C-terminal splaying of the helices, as well as the asymmetry and helix register shift describe unprecedented dynamic excursions of coiled coils. The low stability of APC-55 and divergence from the expected coiled-coil fold support the suggestion that the APC dimerization domain may extend beyond the first 55 residues.     

Designed coiled coils promote folding of a recombinant bacterial collagen

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ～37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ～37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.     

Pitch diversity in α-helical coiled coils

Two complementary methods for measuring local pitch based on heptad position in α-helical coiled coils are described and applied to six crystal structures. The results reveal a diversity of pitch values: two-stranded coiled coils appear to have pitch values near 150 Å the values for three- and four-stranded coiled coils range closer to 200 Å. The methods also provide a rapid and sensitive gauge of local coiled-coil conformation. Polar or charged residues in the apolar interface between coiled-coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled-coil stability. Moreover, the identification of a skip residue (heptad frame shift) in the hemaglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes. These results on relatively short coiled-coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established. We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high-resolution crystal structure—a result not readily discerned by direct comparison of atomic coordinates. Taken together, these methods reveal specific aspects of coiled-coil structure which may escape detection by global analyses of pitch. © 1993 Wiley-Liss, Inc.     

Effects of charged amino acids at b and c heptad positions on specificity and stability of four-chain coiled coils

An understanding of the balance of chemical forces responsible for protein stability and specificity of structure is essential for the success of efforts in protein design. Specifically, electrostatic interactions between charged amino acids have been explored extensively to understand the contribution of this force to protein stability. Much research on the importance of electrostatic interactions as specificity and stability determinants in two-stranded coiled coils has been done, but there remains significant controversy about the magnitude of the attractive forces using such systems. We have developed a four-stranded coiled-coil system with charged residues incorporated at b and c heptad positions to explore the role of charge interactions. Here, we test quantitatively the effects of varying sidechain length on the magnitude of such electrostatic interactions. We synthesized peptides containing either aspartate or ornithine at both b and c heptad positions and tested their ability to self-associate and to hetero-associate with one another and with peptides containing glutamate or lysine at the same positions. We find that interactions between glutamate and either lysine or ornithine are more favorable than the corresponding interactions involving aspartate. In each case, charged interactions provide additional stability to coiled coils, although helix propensity effects may play a significant role in determining the overall stability of these structures.     

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

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.     

Invited review the coiled coil silk of bees, ants, and hornets

In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is α-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

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.