The packing of α-helices: simple coiled-coils

Crick, Francis

doi:10.1107/s0365110x53001964

Cited by 1,610 publications

(1,177 citation statements)

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“…It has been proposed that the association of SNAP-25 with synaptobrevin involves the interaction of ␣-helical domains via formation of intermolecular coiledcoil structures (Chapman et al, 1994;Hayashi et al, 1994). Such ␣-helices are distinguished by a characteristic pattern of hydrophobic residues, which are exposed along one side of the helix, generating an intermolecular hydrophobic core in the twostranded coiled-coil formation (Crick, 1953). Sequence comparison reveals that the periodicity of hydrophobic residues is well preserved in leech SNAP-25, despite the amino acid differences in this domain (Fig.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

et al. 1997

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We have studied the effects of tetanus toxin and botulinus toxin A on neurotransmitter release in the Retzius3 P-cell synapse of the leech and exploited the unique properties of this system, which allow for combined physiological and biochemical analyses in single-cell pairs. The sequences of Hirudo medicinalis synaptobrevin and synaptosomal-associated protein of 25 kDa , deduced by cDNA cloning, are 61 and 55% identical, respectively, to their corresponding mammalian homologs. Whereas Hirudo synaptobrevin is proteolyzed by tetanus toxin, its SNAP-25 isoform is resistant to botulinus toxin A cleavage because of amino acid substitutions within and around the putative cleavage site. In close correlation, microinjection of tetanus toxin into the presynaptic neuron produced a block of transmitter release, whereas botulinus toxin A had no effect on synaptic transmission. Subsequent immunoblotting of single-cell pairs demonstrated directly that the tetanus toxinmediated block of exocytosis is accompanied by cleavage of synaptobrevin in the injected neuron, resulting in the generation of a detectable C-terminal cleavage product. Immunoblotting also confirmed the resistance of SNAP-25 to botulinus toxin A cleavage in vivo. Using recombinant proteins, we show that the N-terminal fragment of synaptobrevin released by tetanus toxin, but not its C-terminal membrane-anchored cleavage product, participates with syntaxin and SNAP-25 in synaptic SNAP receptor (SNARE) ternary complex formation in Hirudo. Our data demonstrate a direct correlation between the inhibition of transmitter release and the ability of the neurotoxin to proteolyze its target protein and support the view that SNARE ternary complex formation is an important step leading to synaptic vesicle exocytosis. Key words: transmitter release; synaptobrevin; VAMP; SNAP-25; SNARE; tetanus toxin; botulinus toxin A; single-cell immunoblotting; Hirudo medicinalisCalcium-regulated exocytosis of synaptic vesicles is the primary means of communication between neurons. A complex of conserved cytosolic proteins known as NSF (N-ethylmaleimidesensitive fusion protein) and SNAP proteins (soluble NSF attachment proteins) is required for various intracellular fusion events in eukaryotic cells (Ferro-Novick and Jahn, 1994;Südhof, 1995;Calakos and Scheller, 1996;Rothman and Wieland, 1996). The search for neuronal membrane receptors (SNAREs, SNAP receptors) for these soluble "fusion" factors led to the identification of the vesicle protein synaptobrevin (VAMP) and the plasma membrane proteins, syntaxin and SNAP-25 (synaptosomal-associated protein of 25 kDa) (Söllner et al., 1993a).Synaptobrevins are class II integral membrane proteins that expose most of their sequence at the cytoplasmic face of secretory vesicles (Südhof, 1995). SNAP-25 is an abundant highly conserved membrane-associated protein (Oyler et al., 1989;Risinger et al., 1993). It is post-translationally modified by palmitoyl side chains that mediate membrane binding (Hess et al., 1992;Veit et al., 1996).Syntaxin is a simi...

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Section: Discussionmentioning

confidence: 99%

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

et al. 1997

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“…The length of the ordered part of Spc42_1 is ∼100 Å, and the diameter of the Spc42_1 dimer is ∼24 Å at its widest point (CR-CR distances are ∼14 Å across the dimer). Residues at the core positions show canonical knobs-into-holes packing (57). Fitting Crick parameters for an idealized coiled coil to residues 67-128 of Spc42_1 gave a superhelical radius (R 0 ) of 4.91 Å, an a-position phase angle (φ) of 0.36 radians, and a superhelical frequency (ω 0 ) of -0.65 radians/amino acid (57, 58) ( Figure 5D).…”

Section: Many Coiled Coils But Few Other Domains Are Predicted Inmentioning

confidence: 99%

Analysis of Coiled-Coil Interactions between Core Proteins of the Spindle Pole Body

et al. 2008

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The spindle pole body (SPB) is a multiprotein complex that organizes microtubules in yeast. Due to its large size and association with the nuclear membrane, little is known about its detailed structure. In particular, although many SPB components and some of the interactions between them have been identified, the molecular details of how most of these interactions occur are not known. The prevalence of predicted coiled-coil regions in SPB proteins suggests that some interactions may occur via coiled coils. Here this hypothesis is supported by biochemical characterization of isolated coiled-coil peptides derived from SPB proteins. Formation of four strongly self-associating coiled-coil complexes from Spc29, Spc42, and Spc72 was demonstrated by circular dichroism (CD) spectroscopy and a fluorescence resonance energy transfer (FRET) assay. Many weaker self-and heteroassociations were also detected by CD, FRET, and/or cross-linking. The thermal stabilities of nine candidate homooligomers were assessed; six unfolded cooperatively with melting temperatures ranging from <11 to >50 °C. Solution studies established that coiled-coil peptides derived from Spc42 and Spc72 form parallel dimers, and this was confirmed for Spc42 by a high-resolution crystal structure. These data contribute to a growing body of knowledge that will ultimately provide a detailed model of the SPB structure.

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“…The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13)(14)(15)(16)(17).…”

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confidence: 99%

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

et al. 2003

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We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 µM (E3/K3). From these K d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.The R-helical coiled-coil is an oligomerization domain that is naturally present in a wide variety of proteins such as transcription factors, cytoskeletal proteins, motor proteins, and viral fusion proteins (1-3). The structural properties of coiled-coils have been extensively characterized from numerous high-resolution crystal and NMR structures (3-5). The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13-17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5, 18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for ...

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The packing of α-helices: simple coiled-coils

Cited by 1,610 publications

References 6 publications

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

Analysis of Coiled-Coil Interactions between Core Proteins of the Spindle Pole Body

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

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