Α-helical Coils Research Articles

Two-Metal Ion, Ni(II) and Cu(II), Binding α-Helical Coiled Coil Peptide

Metalloproteins are an attractive target for de novo design. Usually, natural proteins incorporate two or more (hetero- or homo-) metal ions into their frameworks to perform their functions, but the design of multiple metal-binding sites is usually difficult to achieve. Here, we undertook the de novo engineering of heterometal-binding sites, Ni(II) and Cu(II), into a designed coiled coil structure based on an isoleucine zipper (IZ) peptide. Previously, we described two peptides, IZ-3adH and IZ-3aH. The former has two His residues and forms a triple-stranded coiled coil after binding Ni(II), Zn(II), or Cu(II). The latter has one His residue, which allowed binding with Cu(II) and Zn(II), but not with Ni(II). On the basis of these properties, we newly designed IZ(5)-2a3adH as a heterometal-binding peptide. This peptide can bind Cu(II) and Ni(II) simultaneously in the hydrophobic core of the triple-stranded coiled coil. The first metal ion binding induced the folding of the peptide into the triple-stranded coiled coil, thereby promoting the second metal ion binding. This is the first example of a peptide that can bind two different metal ions. This construction should provide valuable insights for the de novo design of metalloproteins.

The Intermediate Filament Architecture as Determined by X-Ray Diffraction Modeling of Hard α-Keratin

Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard α-keratin intermediate filaments under in vivo conditions. In short, the α-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.

Dimerization of recombinant tobacco mosaic virus movement protein.

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-beta-D-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant alpha-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane alpha-helical coiled coils.

Six-helix bundle assembly and analysis of the central core of mumps virus fusion protein

The fusion protein of enveloped viruses mediates the fusion between the viral and cellular membranes, allowing the penetration of the viral genomes into the host cell. Many of these proteins share a common fold comprising a central core trimer of anti-parallel coiled-coil heterodimers, which are formed by two discontinuous heptad repeat (HR) motifs located at the ectodomain of the fusion proteins. In this study, we constructed and purified the corresponding regions (HR1 and HR2) of mumps virus fusion protein that are predicted to form coiled coil. The HR1 and HR2 were expressed and purified separately or as a single chain connected by an amino acid linker (HR1-linker-HR2, named 2-Helix). Series of biochemical and biophysical analyses of the expressed proteins have shown that HR1 and HR2 of mumps virus fusion protein share the common features of other enveloped virus fusion proteins. CD spectral results show that HR1 forms an α-helical coil structure while HR2 exists as an unstructured monomer in PBS in nature. Mixtures of HR1 and HR2 could form a stable six-helix bundle, indicating the interaction of HR1 and HR2. The 2-Helix protein also shows characteristic properties of the 6-helix bundle. Therefore, mumps virus fusion protein has a common core architecture and its HR regions could be used as a drug target for virus fusion inhibitors.

Reversible Self-Organization of Poly(ethylene glycol)-Based Hybrid Block Copolymers Mediated by a De Novo Four-Stranded α-Helical Coiled Coil Motif

This paper describes the synthesis and supramolecular organization of two novel hybrid diblock copolymers based on poly(ethylene glycol) (PEG) and peptide sequences inspired by the coiled coil protein folding motif. The self-organization of the diblock copolymers is driven by the tendency of the peptide segments to form well-defined tertiary structures. In contrast to conventional amphiphilic block copolymers, whose self-organization is driven by unspecific hydrophobic interactions and leads to polydisperse aggregates, it was anticipated that this approach could allow precise control over the aggregation number in aqueous solution. Circular dichroism and analytical ultracentrifugation experiments indicated that the self-organization properties of the peptide segments are retained upon conjugation of PEG, and discrete, well-defined supramolecular aggregates are formed. No evidence was found for unspecific self-organization of the diblock copolymers to large polydisperse structures, as it is the case for conventional amphiphilic block copolymers. In contrast, the self-organization of the PEG-b-peptide diblock copolymers is described as an equilibrium between unimeric block copolymer molecules and dimeric and tetrameric coiled coil aggregates. The relative amounts of these species depend on concentration, temperature, solvent, and the molecular weight of the PEG block.

Structural characterization of a paramagnetic metal-ion-assembled three-stranded alpha-helical coiled coil.

A helical peptide designed to present an all-leucine core upon folding has been shown to exhibit concentration-dependent helicity and to exist as an ill-defined equilibrium population of oligomers. In marked contrast, an identical peptide covalently modified with a 2,2'-bipyridyl group at the N terminus forms a stable three-stranded parallel coiled coil in the presence of transition metal ions. We have employed paramagnetic Ni(2+) and Co(2+) ions to stabilize the trimeric assembly and to exploit their shift and relaxation properties in NMR structural studies. We find that metal-ion binding and helix-bundle folding are tightly coupled. Surprisingly, the three-helix bundle exhibits a dynamic N-terminal region, and a well-structured C-terminal half. The spectra indicate the presence of a dual conformation for the bundle extending from the N terminus to residue 12. The structure of the two isomeric forms has been ascertained from interpretation of NOEs in the Ni(II) complex and (1)H pseudocontact shifts in the Co(II) complex. Two different facial isomers with distinct susceptibility tensors were identified. The bulky leucine side chain at position 3 in the peptide chain appears to play a role in the conformational variation at the N terminus.

Functional dissection of the global repressor Tup1 in yeast: dominant role of the C-terminal repression domain.

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription. Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms. This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins. We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region. Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13. Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini. The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression. Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression. Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression. The N-terminal region containing potential alpha-helical coiled coils is required for Tup1 oligomerization and association with Cyc8. Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2.

Improving Coiled-coil Stability by Optimizing Ionic Interactions

α-Helical coiled coils are a common protein oligomerization motif stabilized mainly by hydrophobic interactions occurring along the coiled-coil interface. We have recently designed and solved the structure of a two-heptad repeat coiled-coil peptide that is stabilized further by a complex network of inter- and intrahelical salt-bridges in addition to the hydrophobic interactions. Here, we extend and improve the de novo design of this two heptad-repeat peptide by four newly designed peptides characterized by different types of ionic interactions. The contribution of these different types of ionic interactions to coiled-coil stability are analyzed by CD spectroscopy and analytical ultracentrifugation. We show that all peptides are highly α-helical and two of them are 100% dimeric under physiological conditions. Furthermore, we have solved the X-ray structure of the most stable of these peptides and the rational design principles are verified by comparing this structure to the structure of the parent peptide. We show that by combining the most favorable inter- and intrahelical salt-bridge arrangements it is possible to design coiled-coil oligomerization domains with improved stability properties.

The number of amino acid residues mismatches correlates with flow cytometry crossmatching results in high PRA renal patients

Highly sensitized renal transplant candidates present a group at high risk for acute and chronic rejection. The probability of finding compatible donors for these recipients is significantly lower in comparison to those who have low PRA values. As a consequence, these patients spend longer time on the waiting list and become tethered to dialysis. The results of final cross match (XM) are critical for making a decision about whether such a candidate receives an organ or not. The degree of donor and recipient HLA compatibility predicts the results of XM. The goal of this study was to expand a variety of acceptable HLA-AB mismatches (MM) for high PRA kidney recipients using the HLAMATCHMAKER algorithm. This strategy focuses on the fine structural features of HLA polymorphism comprising amino acid residues or triplets (AAT), which are located in α-helical coils of HLA molecules and are available to antibodies. We analyzed serum samples from thirty-nine highly alloimmunized recipients (PRA ⩾ 85%). The level of sensitization was detected using FlowPRA Class I Screening Test. This group of transplant candidates included thirteen recipients who demonstrated negative results of final T/B FCXM and twenty-six, who were FCXM positive. The application of the HLAMATCHMAKER algorithm based on the HLA class I donor and recipient typing allowed us to detect the total number of AATMM as well as the number of immunogenic AAT in both FCXM negative and FCXM positive groups of recipients. Significantly greater numbers of both total and highly immunogenic AATMM have been emerged in the group of FCXM positive patients. Furthermore, the results of this analysis have shown a high degree of probability of positive FCXM if the number of highly immunogenic AATMM was ⩾ 1 (χ 2 = 22.9 Yate’s correction; p = 0.000001). We did not observe overlapping between antibody specificity and permissible HLA-AB MM detected using the HLAMATCHMAKER strategy. Thus, the number of highly immunogenic AATMM can serve as a reliable predictive value for final FCXM results in highly sensitized renal transplant candidates. The HLAMATCHMAKER algorithm appears to be the proper strategy to find donors for high PRA recipients.

Analysis of α-Helical Coiled Coils with the Program TWISTER Reveals a Structural Mechanism for Stutter Compensation

α-Helicalcoiled coils represent a widespread protein structure motif distinguished by a seven-residue periodicity of apolar residues in the primary sequence. A characteristic “knobs-into-holes” packing of these residues into a hydrophobic core results in a superhelical, usually left-handed, rope of two or more α-helices. Such a geometry can be parameterized. For this purpose, a new computer program, TWISTER, was developed. With the three-dimensional coordinates as input, TWISTER uses an original algorithm to determine the local coiled-coil parameters as a function of residue number. In addition, heptad positions are assigned based on structural criteria. It is known that frequently encountered discontinuities in the heptad repeat, such as stutters and skips, can be tolerated within a continuous coiled coil but result in a local distortion of its geometry. This was explored in detail with the help of TWISTER for several two- and three-stranded coiled coils. Depending on the particular protein, stutters were found to be compensated locally by an unwinding of the superhelix, α-helical unwinding, or both. In the first case, there is often a local switch from a left-handed to a right-handed superhelix. In general, the geometrical distortion is confined to about two α-helical turns at either side of the stutter. Furthermore, stutters result in a local increase of the coiled-coil radius.

Temperature-induced conformational changes in amyloid β(1-40) peptide investigated by simultaneous FT-IR microspectroscopy with thermal system

Temperature-dependent secondary structures of the amyloid β(1-40) peptide in the solid state were studied by simultaneous Fourier transform infrared/differential scanning calorimetry (FT-IR/DSC) microspectroscopic system with the heating-cooling-reheating cycle. The result indicates that a thermal transition temperature at 45°C was easily obtained from the three-dimensional plot of the transmission FT-IR spectra as a function of temperature. Furthermore, the thermal-dependent conformational transformations, due to denaturation and aggregation, of solid amyloid β(1-40) were mainly evidenced by reducing the compositions from 37 to 20–24% for α-helical and random coil structures but increasing the components from 27 to 45% for intermolecular β-sheet structures. Thermal-irreversible behavior and a poor thermal stability of solid amyloid β(1-40) were also observed from the poor restoration of the secondary conformational changes in the heated sample.

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 α-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 α-helical coiled-coil domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum is a tightly packed parallel two-stranded α-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 α-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.

Mammalian Reovirus M3 Gene Sequences and Conservation of Coiled-Coil Motifs near the Carboxyl Terminus of the μNS Protein

Nucleotide sequences of the mammalian orthoreovirus (reovirus) type 1 Lang and type 2 Jones M3 gene segments were newly determined. The nucleotide sequence of the reovirus type 3 Dearing M3 segment also was determined to compare with a previously reported M3 sequence for that isolate. Comparisons showed Lang and Dearing M3 to be more closely related than either was to Jones M3, consistent with previous findings for other reovirus gene segments. The μNS protein sequences deduced from each M3 segment were shown to be related in a similar pattern as the respective nucleotide sequences and to contain several regions of greater or less than average variability among the three isolates. Identification of conserved methionine codons near the 5′ ends of the Lang, Jones, and Dearing M3 plus strands lent support to the hypothesis that μNSC, a smaller protein also encoded by M3, arises by translation initiation from a downstream methionine codon within the same open reading frame as μNS. Other analyses of the deduced protein sequences indicated that regions within the carboxyl-terminal third of μNS and μNSC from each isolate have a propensity to form α-helical coiled coils, most likely coiled-coil dimers. The new sequences will augment further studies on μNS and μNSC structure and function.

Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone-rod dystrophy.

Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone-rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic alpha-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation in vitro and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca(2+) sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca(2+) concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone-rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant in vitro and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone-rod degeneration.

Side-Chains Configurations in Coiled Coils Revealed by the 5.15-Å Meridional Reflection on Hard α-Keratin X-Ray Diffraction Patterns

The origin of the 5.15-Å meridional reflection on hard α-keratin X-ray diffraction patterns is discussed in terms of side-chains conformations. We show it to reveal specific configurations of the side chains which are common to all two-stranded α-helical coiled coils. Combining literature data on crystallised coiled coil pieces and molecular dynamics results with our X-ray diffraction pattern simulations, we propose rules for the attribution of χ1torsion angles for coiled coils involved in fibres whose structure cannot be resolved at atomic resolution: in a (a b c d e f g) heptad repeat, a and d residues, respectively, adopt mean t and g+configurations, whereas statistical rules are given for the other residues.

Role of the buried glutamate in the alpha-helical coiled coil domain of the macrophage scavenger receptor.

The macrophage scavenger receptor exhibits a pH-dependent conformational change around the carboxy-terminal half of the alpha-helical coiled coil domain, which has a representative amino acid sequence of a (defgabc)n heptad. We previously demonstrated that a peptide corresponding to this region formed a random coil structure at pH 7 and an alpha-helical coiled coil structure at pH 5 [Suzuki, K., Doi, T., Imanishi, T., Kodama, T., and Tanaka, T. (1997) Biochemistry 36, 15140-15146]. To determine the amino acid responsible for the conformational change, we prepared several peptides in which the acidic amino acids were replaced with neutral amino acids. Analyses of their structures by circular dichroism and sedimentation equilibrium gave the result that the presence of Glu242 at the d position was sufficient to induce the pH-dependent conformational change of the alpha-helical coiled coil domain. Furthermore, we substituted a Glu residue for the Ile residue at the d or a position of a de novo designed peptide (IEKKIEA)4, which forms a highly stable triple-stranded coiled coil. These peptides exhibited a pH-dependent conformational change similar to that of the scavenger receptor. Therefore, we conclude that a buried Glu residue in the hydrophobic core of a triple-stranded coiled coil has the potential to induce the pH-dependent conformational change. This finding makes it possible to elucidate the functions of natural proteins and to create a de novo protein designed to undergo a pH-dependent conformational change.

The role of protonation and metal chelation preferences in defining the properties of mercury-binding coiled coils

To define the delicate interplay between metal chelation, protein folding and function in metalloproteins, a family of de novo-designed peptides was synthesized that self-assemble in aqueous solution to form two and three-stranded α-helical coiled coils. Each peptide contains a single Cys residue at an a or d position of the heptad repeat. Peptide association thus produces a Cys-rich coordination environment that has been used to bind Hg(II) ions. These peptides display a pH-dependent association, with trimers observed above the pKa of Glu side-chains and dimers below this value. Finite-difference Poisson-Boltzmann calculations suggest that the dimeric state decreases the unfavorable electrostatic interactions between positively charged Lys side-chains (relative to the trimer). The Cys-containing peptides bind Hg(II) in a position-dependent fashion. Cys at a positions form three-coordinate Hg complexes at high pH where the trimeric aggregation state predominates, and two-coordinate complexes at lower pH. A d position Cys, however, is only able to generate the two-coordinate complex, illustrating the difference in coordination geometry between the two positions in the coiled coil. The binding of Hg(II) was also shown to substantially increase the stability of the helical aggregates.

The C-terminal Domain of Matrilin-2 Assembles into a Three-stranded α-Helical Coiled Coil

Matrilin-2 is a member of von Willebrand factor A containing extracellular matrix proteins in which the cDNA-derived sequence shows similar domain organization to cartilage matrix protein/matrilin-1, but information on the protein structure is limited. Here we studied the oligomerization potential of a synthetic peptide NH2-ENLILFQNVANEEVRKLTQRLEEMTQRMEALENRLKYR-COOH corresponding to the C-terminal sequence of mouse matrilin-2. The central portion of this sequence shows a periodicity of hydrophobic residues occupying positions a and d of a heptad pattern (abcdefg)n, which is characteristic for alpha-helical coiled-coil proteins. Circular dichroism spectroscopy revealed a high alpha-helical content, and the shape of the spectra is indicative for a coiled-coil conformation. Chemical cross-linking and size exclusion chromatography suggest a homotrimeric configuration. Thermal denaturation in benign buffer shows a single cooperative transition with DeltaH0 = -375 kJ/mol. Melting temperatures Tm varied from 38 to 51 degreesC within a concentration range of 10 to 85 microM, which is about 35 degreesC lower than determined for a peptide corresponding to the C-terminal domain of matrilin-1. The data suggest that despite the low sequence identity within this region, matrilin-2 will form a homotrimer as matrilin-1 does.

Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: potential role for coiled coil interaction.

Troponin T (TnT), a thin filament myofibrillar protein, is essential for the Ca2+ regulation of striated muscle contraction in vertebrates, both in vivo and in vitro. To understand the role of TnT in this process, its interaction with two other troponin components, troponin I (TnI) and troponin C (TnC) was examined by using the yeast two hybrid system, which is a genetic approach to detect protein-protein interactions. Computer assisted analysis of phylogenetically distant TnT amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential for alpha-helical coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TnI sequences. These protein motifs appeared to be the regions where TnI-TnT interaction may take place. Deletions and point mutations in TnT, which disrupted its HR motif, severely reduced or abolished TnI binding, but binding to TnC was not affected, indicating that the TnT-TnI and TnT-TnC binary interactions can be uncoupled. Remarkably, the truncated fragments of TnT and TnI in which the HR motifs were retained showed binary interaction in the yeast two hybrid system. It was also observed that the formation of the TnT-TnI heterodimers is favored over the homodimers TnT-TnT and TnI-TnI. These results indicate that the evolutionarily conserved HR motifs may play a role in TnT-TnI dimerization, presumably through the formation of alpha-helical coiled coils.

Why Fibrous Proteins Are Romantic

Here I give a personal account of the great history of fibrous protein structure. I describe how Astbury first recognized the essential simplicity of fibrous proteins and their paradigmatic role in protein structure. The poor diffraction patterns yielded by these proteins were then deciphered by Pauling, Crick, Ramachandran and others (in part by model building) to reveal α-helical coiled coils, β-sheets, and the collagen triple helical coiled coil—all characterized by different local sequence periodicities. Longer-range sequence periodicities (or “magic numbers”) present in diverse fibrous proteins, such as collagen, tropomyosin, paramyosin, myosin, and were then shown to account for the characteristic axial repeats observed in filaments of these proteins. More recently, analysis of fibrous protein structure has been extended in many cases to atomic resolution, and some systems, such as “leucine zippers,” are providing a deeper understanding of protein design than similar studies of globular proteins. In the last sections, I provide some dramatic examples of fibrous protein dynamics. One example is the so-called “spring-loaded” mechanism for viral fusion by the hemagglutinin protein of influenza. Another is the possible conformational changes in prion proteins, implicated in “mad cow disease,” which may be related to similar transitions in a variety of globular and fibrous proteins.