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.     

Deriving how far structural information is transmitted through parallel homodimeric coiled‐coils: A correlation analysis of helical staggers

How local conformation is affected by local sequence is fairly well understood for alpha‐helical coiled‐coils, but less is known about how local conformation is influenced by distant features. Here, I describe an approach to detect such an effect, based on computing correlation coefficients of local out‐of‐register alignments, or so‐called “staggers” between the helices, as a function of the axial distance between the staggers. This approach requires parallel homodimers, in which each stagger can occur with two “signs,” where either one helix or the other is shifted towards the N terminus. The signs of such staggers separated by up to 12 residues are strongly correlated, indicating that the conformations of the ends of coiled‐coils are commonly influenced by attached structures. Thus, the structures of coiled‐coil residues aberrantly attached to alternative proteins, such as those resulting from leukemogenic chromosomal rearrangements, may be distinguishable from those in normal tissues, and in turn serve as targets of selective drug design. The signs of helical staggers separated by between 13 and 30 residues are moderately yet significantly correlated, indicating that some of the coiled‐coils transmit this conformational feature axially for at least 45 Å. A positive, albeit noisy, correlation also exists among tropomyosin coiled‐coils for signed staggers separated by the 40‐residue actin repeat distance, consistent with the semi‐flexible tropomyosin filament binding F‐actin and regulating skeletal muscle contraction in a partially cooperative manner. Communication of the signs of axial staggers is explained in part by minimization of main‐chain hydrogen bond deformations. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Tropomyosin crystal structure and muscle regulation

The crystal structure of tropomyosin filaments has been solved to 15 A resolution by refinement of models against the diffraction data and heavy atom labeling of cysteine residues. These results confirm and extend earlier findings. The improved maps reveal the pitch of the coiled coil, the location of the cysteine residues, and the location and features of the overlapping molecular ends in the filaments. A correlation can now be made between regions of the amino acid sequence and key features of the molecule, such as contact sites in the lattice and departures from regularity along the coiled coil. The crystal shows remarkable dynamic features and the relative flexibility of different parts of the molecule as well as its anisotropic character have been determined. The structure and motions of tropomyosin in the crystal provide information on the structure of tropomyosin in muscle and its possible role in regulation. An atomic model of the molecule has been constructed, based on the low resolution X-ray results, together with the stereochemistry of alpha-helical coiled coils. In contrast to previous views, the molecule appears to display but one set of seven alpha-sites that permit weak linkages of the flexible tropomyosin filament to the actin helix. Correspondingly, we picture that in the "off" state of ATPase activity, the alpha-sites are not occupied; in the "on" state, they are only partly occupied; and in the "potentiated" state, they are more completely saturated. Control of contraction is therefore seen as a statistical mechanism requiring at least three distinct average conformations for the tropomyosin molecule on the actin helix.     

Influence of a heptad repeat stutter on the pH‐dependent conformational behavior of the central coiled‐coil from influenza hemagglutinin HA2

The coiled‐coil is one of the most common protein structural motifs. Amino acid sequences of regions that participate in coiled‐coils contain a heptad repeat in which every third then forth residue is occupied by a hydrophobic residue. Here we examine the consequences of a “stutter,” a deviation of the idealized heptad repeat that is found in the central coiled‐coil of influenza hemagluttinin HA2. Characterization of a peptide containing the native stutter‐containing HA2 sequence, as well as several variants in which the stutter was engineered out to restore an idealized heptad repeat pattern, revealed that the stutter is important for allowing coiled‐coil formation in the WT HA2 at both neutral and low pH (7.1 and 4.5). By contrast, all variants that contained idealized heptad repeats exhibited marked pH‐dependent coiled‐coil formation with structures forming much more stably at low pH. A crystal structure of one variant containing an idealized heptad repeat, and comparison to the WT HA2 structure, suggest that the stutter distorts the optimal interhelical core packing arrangement, resulting in unwinding of the coiled‐coil superhelix. Interactions between acidic side chains, in particular E69 and E74 (present in all peptides studied), are suggested to play a role in mediating these pH‐dependent conformational effects. This conclusion is partially supported by studies on HA2 variant peptides in which these positions were altered to aspartic acid. These results provide new insight into the structural role of the heptad repeat stutter in HA2. Proteins 2014; 82:2220–2228. © 2014 Wiley Periodicals, Inc.     

Crystal structure of tropomyosin at 7 Angstroms resolution

Tropomyosin is a 400A-long coiled coil that polymerizes to form a continuous filament that associates with actin in muscle and numerous non-muscle cells. Tropomyosin and troponin together form a calcium-sensitive switch that is responsible for thin-filament regulation of striated muscle. Subtle structural features of the molecule, including non-canonical aspects of its coiled-coil motif, undoubtedly influence its association with f-actin and its role in thin filament regulation. Previously, careful inspection of native diffraction intensities was sufficient to construct a model of tropomyosin at 9A resolution in a spermine-induced crystal form that diffracts anisotropically to 4A resolution. Single isomorphous replacement (SIR) phasing has now provided an empirical determination of the structure at 7A resolution. A novel method of heavy-atom analysis was used to overcome difficulties in interpretation of extremely anisotropic diffraction. The packing arrangement of the molecules in the crystal, and important aspects of the tropomyosin geometry such as non-uniformities of the pitch and variable bending and radius of the coiled coil are evident.     

A Peek into Tropomyosin Binding and Unfolding on the Actin Filament

Background

Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin.

Principal Findings

Tropomyosin''s periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle α-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism).

Conclusions

This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.     

Structural destabilization of tropomyosin induced by the cardiomyopathy‐linked mutation R21H

The missense mutation R21H in striated muscle tropomyosin is associated with hypertrophic cardiomyopathy, a genetic cardiac disease and a leading cause of sudden cardiac death in young people. Tropomyosin adopts conformation of a coiled coil which is critical for regulation of muscle contraction. In this study, we investigated the effects of the R21H mutation on the coiled‐coil structure of tropomyosin and its interactions with its binding partners, tropomodulin and leiomodin. Using circular dichroism and isothermal titration calorimetry, we found that the mutation profoundly destabilized the structural integrity of αTM1a_1‐28Zip, a chimeric peptide containing the first 28 residues of tropomyosin. The mutated αTM1a_1‐28Zip was still able to interact with tropomodulin and leiomodin. However, the mutation resulted in a ～30‐fold decrease of αTM1a_1‐28Zip's binding affinity to leiomodin. We used a crystal structure of αTM1a_1‐28Zip that we solved at 1.5 Å resolution to study the mutation's effect in silico by means of molecular dynamics simulation. The simulation data indicated that while the mutation disrupted αTM1a_1‐28Zip's coiled‐coil structure, most notably from residue Ala18 to residue His31, it may not affect the N‐terminal end of tropomyosin. The drastic decrease of αTM1a_1‐28Zip's affinity to leiomodin caused by the mutation may lead to changes in the dynamics at the pointed end of thin filaments. Therefore, the R21H mutation is likely interfering with the regulation of the normal thin filament length essential for proper muscle contraction.     

Motions of tropomyosin. Crystal as metaphor.

Movements of tropomyosin play an essential role in muscle regulation. This fibrous protein is a two-chain alpha-helical coiled coil that bonds head to tail to form cables wound in the two long grooves of the actin helix. The regulatory switch consists of tropomyosin and a "globular" Ca2+-sensitive protein complex called troponin. The structure of the tropomyosin filaments has now been determined by x-ray crystallography to approximately 15 A resolution. The complete sequence of alpha-tropomyosin is known; by using mercury markers on the cysteine residues the ends of the molecules in the filaments have been identified. Details of the coiled-coil structure have also been visualized by refinement of models against the diffraction data. The average pitch of the coiled coil is approximately 137 A, so that each tropomyosin molecule can make similar contacts with seven actin monomers. The electron density map also indicates that departures from the alpha-helical coiled coil occur in a few localized regions of the molecule, especially at the overlapping ends. Motions of tropomyosin in the crystal lattice are displaced by the character of the Bragg reflections and the strong diffuse scatter. These effects depend markedly on temperature. It appears that the molecular filaments fluctuate freely in a direction perpendicular to their axes. Moreover, the C-terminal half of the molecule "unfolds" to some degree at less than physiological temperatures. Crystallographic results on co-crystals of tropomyosin and a component of troponin (TnT) suggest that this subunit consists of structurally distinct domains, so that the troponin complex is not in fact simply "globular". The interactions of the extended alpha-helical region of TnT may "stiffen" tropomyosin and influence its motions. We picture the tropomyosin/troponin switch in muscle as a restless cable, perpetually making and breaking bonds as it vibrates on the thin filament. These movements of tropomyosin probably depend on two aspects of its design: the regular pattern of coiled-coil linkages with actin; and the aperiodic features that allow flexibility and motion.     

Generalized Crick equations for modeling noncanonical coiled coils

Crick envisaged the alpha-helical coiled coil to result from systematic bending of an alpha-helix such that every seventh residue was structurally equivalent, and he derived equations for the coordinates of the backbone atoms. Crick's predictions were vindicated experimentally and coiled-coil sequences were shown to have hydrophobic residues alternately spaced 3 and 4 residues apart. Nonetheless, in some coiled coils such canonical heptad repeats are interrupted by inserts of 3 or 4 residues generating decad and hendecad motifs. The supercoiling of the coiled coils varies with the sequence pattern, being left- or right-handed in purely heptad-based or hendecad-based motifs, respectively. To model coiled coils with a mixture of motifs, we describe how Crick's equations can be modified for cases where the pitch is not constant. Using the analogy of the bending of a beam, we took the tilt angle to change linearly with distance along the major helix and the pitch of a motif to be affected by neighboring motifs depending on the rigidity of the alpha-helical strands. We tested our approach by fitting the two-, three-, and four-stranded noncanonical coiled coils of GrpE, hemagglutinin, and tetrabrachion. The backbone atoms of the model and crystal structures agreed with root mean square deviations of <1.1 A.     

Structural stability of short subsequences of the tropomyosin chain

The native tropomyosin molecule is a parallel, registered, α-helical coiled coil made from two 284-residiic chains. Long excised subsequences (≥ 95 residues) form the same structure with comparable thermal stability. Here, we investigate local stability using shorter subsequences (20-50 residues) that are chemically synthesized or excised from various regions along the protein chain. Thermal unfolding studies of such shorter peptides by CD in the same solvent medium used in extant studies of the parent protein indicate very low helix content, almost no coiled-coil formation, and high thermal lability of such secondary structure as does form. This behavior is in stark contrast to extant data on leucine-zipper peptides and short “designed” synthetic peptides, many of which have high α-helix content and form highly stable coiled coils. The existence of short coiled coils calls into question the older idea that short subsequences of a protein have little structure. The present study supports the older view, at least in its application to tropomyosin. The intrinsic local α-helical propensity and helix–helix interaction in this prototypical α-helical protein is sufficiently weak as to require not only dimerization, but macro-molecular amplification in order to attain its native conformation in common benign media near neutral pH. © 1995 John Wiley & Sons, Inc.     

The crystal structure of the designed trimeric coiled coil coil-VaLd: implications for engineering crystals and supramolecular assemblies.

The three-dimensional structure of the 29-residue designed coiled coil having the amino acid sequence acetyl-E VEALEKK VAALESK VQALEKK VEALEHG-amide has been determined and refined to a crystallographic R-factor of 21.4% for all data from 10-A to 2.1-A resolution. This molecule is called coil-VaLd because it contains valine in the a heptad positions and leucine in the d heptad positions. In the trigonal crystal, three molecules, related by a crystallographic threefold axis, form a parallel three-helix bundle. The bundles are stacked head-to-tail to form a continuous coiled coil along the c-direction of the crystal. The contacts among the three helices within the coiled coil are mainly hydrophobic: four layers of valine residues alternate with four layers of leucine residues to form the core of the bundle. In contrast, mostly hydrophilic contacts mediate the interaction between trimers: here a total of two direct protein--protein hydrogen bonds are found. Based on the structure, we propose a scheme for designing crystals of peptides containing continuous two-, three-, and four-stranded coiled coils.     

Instability in a coiled‐coil signaling helix is conserved for signal transduction in soluble guanylyl cyclase

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC‐1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N‐terminus in sGC activates cyclase activity near the C‐terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small‐molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled‐coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad‐repeating pattern for coiled‐coil motifs, but also invariant positions that disfavor coiled‐coil stability. Full‐length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled‐coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.     

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.     

Crystal structure of a super leucine zipper,an extended two‐stranded super long coiled coil

Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 Å resolution. The peptide monomer shows a helix trunk with short curved N‐ and C‐termini. In the crystal, two monomers cross in 35° and form an X‐shaped dimer, and each X‐shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two‐stranded, parallel, super long coiled coil rather than a discrete, two‐helix coiled coil of the wild‐type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild‐type leucine zipper, the N‐terminus of the mutant has a dramatic conformational change and the C‐terminus has one more residue Glu 32 determined. The mutant X‐shaped dimer has a large crossing angle of 35° instead of 18° in the wild‐type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self‐assembling protein fibers.     

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.     

Coiled‐coils: The long and short of it

Coiled‐coils are found in proteins throughout all three kingdoms of life. Coiled‐coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled‐coil. Other coiled‐coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled‐coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled‐coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled‐coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled‐coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.     

Dual requirement for flexibility and specificity for binding of the coiled-coil tropomyosin to its target, actin

The coiled coil is a widespread motif involved in oligomerization and protein-protein interactions, but the structural requirements for binding to target proteins are poorly understood. To address this question, we measured binding of tropomyosin, the prototype coiled coil, to actin as a model system. Tropomyosin binds to the actin filament and cooperatively regulates its function. Our results support the hypothesis that coiled-coil domains that bind to other proteins are flexible. We made mutations that alter interface packing and stability as well as mutations in surface residues in a postulated actin binding site. Actin affinity, measured by cosedimentation, was correlated with coiled-coil stability and local instability and side chain flexibility, analyzed with circular dichroism and fluorescence spectroscopy. The flexibility from interruptions in the stable coiled-coil interface is essential for actin binding. The surface residues in a postulated actin binding site participate in actin binding when the coiled coil within it is poorly packed.     

Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer

The crystal structure of a polypeptide chain fragment from the surface layer protein tetrabrachion from Staphylothermus marinus has been determined at 1.8 A resolution. As proposed on the basis of the presence of 11-residue repeats, the polypeptide chain fragment forms a parallel right-handed coiled coil structure. Complementary hydrophobic interactions and complex networks of surface salt bridges result in an extremely thermostable tetrameric structure with remarkable properties. In marked contrast to left-handed coiled coil tetramers, the right-handed coiled coil reveals large hydrophobic cavities that are filled with water molecules. As a consequence, the packing of the hydrophobic core differs markedly from that of a right-handed parallel coiled coil tetramer that was designed on the basis of left-handed coiled coil structures.