The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils.

The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.     

Review: conformation and folding of novel beta-structural elements in viral fiber proteins: the triple beta-spiral and triple beta-helix

Apart from alpha-helical coiled coils and the collagen triple helices, fibrous proteins can contain beta-structure in various conformations. Elongated enzymes such as pectate lyase and the bacteriophage P22 tailspike protein contain single-stranded beta-helices. Virus and bacteriophage fibers, which are often trimeric, have been shown to contain novel triple-stranded beta-structures such as the triple beta-spiral and the triple beta-helix. The conformation and folding of viral fibers containing beta-structure are discussed.     

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.     

Designed coiled-coil proteins: synthesis and spectroscopy of two 78-residue alpha-helical dimers.

Receptor-adhesive modular proteins are nongenetic proteins designed to contain ligand, spacer, coil, and linker modules and to interact strongly with integrins or other types of cell-surface receptors. We have designed, chemically synthesized, and characterized a 39-residue peptide chain having a 6-residue ligand module (Gly-Arg-Gly-Asp-Ser-Pro-) for adherence to Arg-Gly-Asp-binding integrin receptors, a 3-residue spacer module (-Gly-Tyr-Gly-) for flexibility, and a 30-residue coil module [-(Arg-Ile-Glu-Ala-Ile-Glu-Ala) 4-Arg-Cys-NH2] containing four 7-residue repeats for dimerization. This chain was designed to form a 78-residue noncovalent dimer (P39) by folding the coils of two chains into an alpha-helical coiled coil through hydrophobic interaction of eight pairs of Ile residues. Air oxidation of P39 gave P78, a 78-residue covalent dimer having a disulfide bridge linking its C termini. Raman spectroscopy indicated that both synthetic proteins have high alpha-helical content. Ultraviolet circular dichroic spectroscopy indicated that both dimers contain stable alpha-helical coiled coils. Its C-terminal disulfide bridge renders P78 significantly more stable than P39 to thermal denaturation or denaturation by urea. The coiled coil of P39 was 30% unfolded near 55 degrees C and half-unfolded in 8 M urea, while that of P78 was 30% unfolded only near 85 degrees C. These studies have demonstrated the feasibility of using these ligand, spacer, and coil modules to construct the designed coiled-coil proteins P39 and P78, a stage in the nanometric engineering of receptor-adhesive modular proteins.     

Fifty years of coiled-coils and alpha-helical bundles: a close relationship between sequence and structure

alpha-Helical coiled coils are remarkable for the diversity of related conformations that they adopt in both fibrous and globular proteins, and for the range of functions that they exhibit. The coiled coils are based on a heptad (7-residue), hendecad (11-residue) or a related quasi-repeat of apolar residues in the sequences of the alpha-helical regions involved. Most of these, however, display one or more sequence discontinuities known as stutters or stammers. The resulting coiled coils vary in length, in the number of chains participating, in the relative polarity of the contributing alpha-helical regions (parallel or antiparallel), and in the pitch length and handedness of the supercoil (left- or right-handed). Functionally, the concept that a coiled coil can act only as a static rod is no longer valid, and the range of roles that these structures have now been shown to exhibit has expanded rapidly in recent years. An important development has been the recognition that the delightful simplicity that exists between sequence and structure, and between structure and function, allows coiled coils with specialized features to be designed de novo.     

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.     

Translated products of tandem microgene repeats exhibit diverse properties also seen in natural proteins

Repetitiousness is often observed in the primary and tertiary structures of proteins. We are intrigued by the potential role played by periodicity in the evolution of proteins and have created artificial repetitious proteins from repeats of short DNA sequences (microgenes). In this paper we characterize the physicochemical properties of six such artificially created proteins, which are the translated products of repeats of three microgenes. Three of the six proteins contain beta-sheet-like structures and are rather hydrophobic in nature. These proteins form macroscopic membranous structures in the presence of monovalent cationic ions, suggesting they have the capacity to promote strong intermolecular interactions. Of the other three proteins, one is comprised of alpha-helices and two have disordered structures. Small angle X-ray scattering analysis indicates that the artificial proteins do not fold as tightly as natural proteins, but are more compact than if completely denatured. One alpha-helical protein whose microgene unit was designed from coiled coil proteins was crystallized, demonstrating that repetitious artificial proteins can undergo transition to a more ordered state under appropriate conditions. Application of this approach to the development of a novel protein engineering system is discussed.     

The coiled-coil trigger site of the rod domain of cortexillin I unveils a distinct network of interhelical and intrahelical salt bridges

BACKGROUND: The parallel two-stranded alpha-helical coiled coil is the most frequently encountered subunit-oligomerization motif in proteins. The simplicity and regularity of this motif have made it an attractive system to explore some of the fundamental principles of protein folding and stability and to test the principles of de novo design. RESULTS: The X-ray crystal structure of the 18-heptad-repeat alpha-helical coiled-coil domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum is a tightly packed parallel two-stranded alpha-helical coiled coil. It harbors a distinct 14-residue sequence motif that is essential for coiled-coil formation, and is a prerequisite for the assembly of cortexillin I. The atomic structure reveals novel types of ionic coiled-coil interactions. In particular, the structure shows that a characteristic interhelical and intrahelical salt-bridge pattern, in combination with the hydrophobic interactions occurring at the dimer interface, is the key structural feature of its coiled-coil trigger site. CONCLUSIONS: The knowledge gained from the structure could be used in the de novo design of alpha-helical coiled coils for applications such as two-stage drug targeting and delivery systems, and in the design of coiled coils as templates for combinatorial helical libraries in drug discovery and as synthetic carrier molecules.     

New aspects of the alpha-helix to beta-sheet transition in stretched hard alpha-keratin fibers

The putative transformation of alpha-helices into beta-sheets has been studied for more than 50 years in the case of hard alpha-keratin. In a previous study of stretched keratin fibers, we specified the conditions for beta-sheet appearance within horsehair: the formation of beta-sheets requires at least 30% relative humidity. However, this phenomenon was observed in the whole tissue. Then there was no clear chemical identification of the beta-sheets (keratin or matrix proteins) and the exact location of the beta-sheets across the fiber could not be specified. In this study, using wide-angle x-ray scattering and high spatial resolution infrared microspectroscopy, we could determine and characterize the structural elements across hair sections stretched in water, which provides new information about the aforementioned transition. Our results show that the process can be split into three steps: 1), unraveling of the alpha-helical coiled-coil domains, which starts at roughly 5% macroscopic strain; 2), further transformation of the unraveled coiled-coils into beta-sheet structures, which occurs above roughly 20% macroscopic strain; and 3), spatial expanding of the beta-structured zones from the sample center to its periphery.     

Phylogenetic occurrence of coiled coil proteins: Implications for tissue structure in metazoa via a coiled coil tissue matrix

We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained “extended” (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1–2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in Metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.     

A procedure for refining a coiled coil protein structure using x-ray fiber diffraction and modeling

We describe a combined use of experimental and simulation techniques to configure side chains in a coiled coil structure. As already demonstrated in a previous work, x-ray diffraction patterns from hard alpha-keratin fibers in the 5.15 A meridian zone reflect the global configuration of the chi(1) dihedral angle of the coiled coil side chains. Molecular simulations, such as energy minimization and molecular dynamics, and rotameric representation in the PDB, are used here on a heterodimeric coiled coil to investigate the dihedral angle distribution along the sequence. Different procedures have been used to build the structure, the quality assessment was based on the agreement between the simulated diffraction patterns and the experimental ones in the fingerprint region of coiled coils (5.15 A). The best one for building a realistic coiled coil structure consists of placing the side chains using molecular dynamics (MD) simulations, followed by side chain positioning using SMD or SCWRL procedures. The side chains and the backbone are equilibrated during the MD until they reach an equilibrium state for the t/g(+) ratio. Positioning the side chains on the resulting backbone, using the above procedures, gives rise to a well-defined 5.15 A meridian reflection.     

Conservation of essential design features in coiled coil silks

Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended beta-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small ( approximately 30 kDa each) and nonrepetitive and adopt a coiled coil structure. We examined silks from the 3 superfamilies of the Aculeata (Hymenoptera: Apocrita) by infrared spectroscopy and found coiled coil structure in bees (Apoidea) and in ants (Vespoidea) but not in parasitic wasps of the Chrysidoidea. We subsequently identified and sequenced the silk genes of bumblebees, bulldog ants, and weaver ants and compared these with honeybee silk genes. Each species produced orthologues of the 4 small fibroin proteins identified in honeybee silk. Each fibroin contained a continuous predicted coiled coil region of around 210 residues, flanked by 23-160 residue length N- and C-termini. The cores of the coiled coils were unusually rich in alanine. There was extensive sequence divergence among the bee and ant silk genes (<50% similarity between the alignable regions of bee and ant sequences), consistent with constant and equivalent divergence since the bee/ant split (estimated to be 155 Myr). Despite a high background level of sequence diversity, we have identified conserved design elements that we propose are essential to the assembly and function of coiled coil silks.     

Signal transduction: hair brains in bacterial chemotaxis

The conserved cytoplasmic domains of bacterial chemotaxis receptors are a fibrous arrangement of alpha-helical coiled coils that look a lot like hair. Such bundles of alpha-helical filaments mediate sensory-motor responses in all prokaryotic cells. How do they work? Very nearly perfectly is probably as good an answer as any.     

Kinking the coiled coil--negatively charged residues at the coiled-coil interface

The coiled coil is one of the most common protein-structure motifs. It is believed to be adopted by 3-5% of all amino acids in proteins. It comprises two or more alpha-helical chains wrapped around one another. The sequences of most coiled coils are characterized by a seven-residue (heptad) repeat, denoted (abcdefg)(n). Residues at the a and d positions define the helical interface (core) and are usually hydrophobic, though about 20% are polar or charged. We show that parallel coiled-coils have a unique pattern of their negatively charged residues at the core positions: aspartic acid is excluded from these positions while glutamic acid is not. In contrast the antiparallel structures are more permissive in their amino acid usage. We show further, and for the first time, that incorporation of Asp but not Glu into the a positions of a parallel coiled coil creates a flexible hinge and that the maximal hinge angle is being directly related to the number of incorporated mutations. These new computational and experimental observations will be of use in improving protein-structure predictions, and as rules to guide rational design of novel coiled-coil motifs and coiled coil-based materials.     

Evidence that the stalk of Drosophila kinesin heavy chain is an alpha- helical coiled coil

Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.     

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."     

Segmented alpha-helical coiled-coil structure of the protein giardin from the Giardia cytoskeleton

The parasitic flagellate Giardia is the source of a filamentous protein, giardin, which binds to microtubules. The primary sequence of one giardin chain has been decoded from the base sequences of cDNAs isolated by antibody screens of a library constructed in the expression vector lambda gt11. The amino acid sequence favours a continuous alpha-helical fold for the protein without any inserts of a non-helical character. Analysis of apolar residue positions revealed 35 repeating heptads consistent with coiled-coil structure. This conformation relates giardin to the alpha-type fibrous proteins (k-m-e-f class) like tropomyosin and myosin (also found in Giardia). The giardin sequence has a regular series of skip residues like those at certain positions in the rod section of nematode myosin where the internal apolar seam of the coiled coil is shifted on the helix surface. The skips divide the giardin coil into quasi-equivalent structural segments about 4 nm in length, which might be domains for combining with tubulin subunits in the microtubule surface lattice.