A distinct 14 residue site triggers coiled-coil formation in cortexillin I
1998; Springer Nature; Volume: 17; Issue: 7 Linguagem: Inglês
10.1093/emboj/17.7.1883
ISSN1460-2075
AutoresMichel O. Steinmetz, Alexander Stock, Therese Schulthess, Ruth Landwehr, Ariel Lustig, Jan Faix, Günther Gerisch, Ueli Aebi, Richard A. Kammerer,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoArticle1 April 1998free access A distinct 14 residue site triggers coiled-coil formation in cortexillin I Michel O. Steinmetz Michel O. Steinmetz M.E.Müller Institute for Microscopy Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Alexander Stock Alexander Stock Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Therese Schulthess Therese Schulthess Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Ruth Landwehr Ruth Landwehr Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Ariel Lustig Ariel Lustig Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Jan Faix Jan Faix Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Günther Gerisch Günther Gerisch Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Ueli Aebi Corresponding Author Ueli Aebi M.E.Müller Institute for Microscopy Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Richard A. Kammerer Richard A. Kammerer Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Michel O. Steinmetz Michel O. Steinmetz M.E.Müller Institute for Microscopy Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Alexander Stock Alexander Stock Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Therese Schulthess Therese Schulthess Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Ruth Landwehr Ruth Landwehr Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Ariel Lustig Ariel Lustig Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Jan Faix Jan Faix Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Günther Gerisch Günther Gerisch Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany Search for more papers by this author Ueli Aebi Corresponding Author Ueli Aebi M.E.Müller Institute for Microscopy Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Richard A. Kammerer Richard A. Kammerer Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Author Information Michel O. Steinmetz1, Alexander Stock2, Therese Schulthess3, Ruth Landwehr3, Ariel Lustig3, Jan Faix2, Günther Gerisch2, Ueli Aebi 1 and Richard A. Kammerer3 1M.E.Müller Institute for Microscopy Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland 2Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany 3Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1883-1891https://doi.org/10.1093/emboj/17.7.1883 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311–Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311–Asp324 yielded ∼30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311–Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I. Introduction Subunit oligomerization of many proteins is mediated by coiled-coil domains (Lupas, 1996; Kammerer, 1997; Kohn et al., 1997). Typically, coiled coils consist of two to five right-handed amphipatic α-helices which coil around each other to form a slightly left-handed supercoil. Polypeptide segments giving rise to coiled coils are characterized by so-called heptad repeats of seven amino acid residues, denoted a to g (Sodek et al., 1972; McLachlan and Stewart, 1975; Cohen and Parry, 1990). The residues at positions a and d are mostly apolar, thereby forming a 3,4-hydrophobic repeat with charged residues occurring frequently at the e and g positions. The hallmark of coiled coils is the distinctive packing of amino acid side chains in the hydrophobic core of the helix bundles, called 'knobs-into-holes' packing, which was first described by Crick (1953). Accordingly, hydrophobic interactions appear to represent the major driving force in stabilizing a coiled coil (Harbury et al., 1993). Ionic interactions between the side chains of neighboring helices are considered relevant to the stability, orientation and stoichiometry of coiled coils (O'Shea et al., 1992; Monera et al., 1994; Zhou et al., 1994; Beck et al., 1997). The simplicity and regularity of the coiled-coil structural motif have made it an attractive system for exploring some of the fundamental features of protein folding and protein–protein interactions. Moreover, coiled coils have been the focus of de novo protein design, a research area which has recently become important in attempts to rationally design multi-stranded coiled coils for a variety of purposes, including medical applications such as the targeting and delivery of drugs by heterodimerization technology, the engineering of synthetic biosensors and carrier molecules, and the discovery of new drugs (for a review see Hodges, 1996). Although the parameters which determine the state of oligomerization and the stability of coiled coils are well known, only limited information is currently available on the mechanistic details leading to the formation of this apparently simple structural motif. Studies on the folding of coiled coils have primarily focused on tropomyosin, myosin and leucine zipper peptides, all producing two-helix structures. Thermodynamic and kinetic studies with leucine zippers suggested that their unfolding follows a simple two-state mechanism (Thompson et al., 1993; Wendt et al., 1995, 1997; Sosnick et al., 1996). In contrast, the unfolding of the much longer coiled-coil domains of tropomyosin (Lehrer, 1978) and skeletal muscle myosin (King et al., 1995) were reported to be non-two-state reactions indicating that these coiled coils may consist of discrete, independently stable subdomains. Kinetic studies using stopped-flow techniques with tropomyosin (Mo et al., 1991, 1992), the leucine zipper domain of the yeast transcriptional activator GCN4 (a 33 residue peptide denoted GCN4-p1; Zitzewitz et al., 1995; Sosnick et al., 1996), and other leucine zipper peptides (Wendt et al., 1995, 1997) suggested a fast bimolecular association step. However, none of these investigations established the mechanistic details underlying the folding pathway of coiled coils which in turn would not only be of theoretical but also, as mentioned above, of considerable practical value. In this context, a rather puzzling, frequently made observation is that relatively long heptad-repeat-containing polypeptide chain fragments derived from stable coiled-coil domains fail to associate into coiled-coil structures. For example, Trybus et al. (1997) have recently reported that smooth muscle myosin fragments with as many as 15 heptad repeats of the coiled-coil rod sequence failed to dimerize. This failure cannot be simply explained by instability due to the type of residues occupying the a and d positions of the heptad repeats, or by electrostatic repulsion of the two chains. As it is not known what factors determine chain association, the failure of chain assembly raises the question of whether there exist distinct sites within heptad-repeat-containing amino acid sequences which are necessary to mediate or initiate coiled-coil formation. We have addressed this question in detail by using the two-stranded coiled-coil oligomerization domain of cortexillin I (Ir), an actin cross-linking protein from Dictyostelium discoideum (Faix et al., 1996). For this purpose, we produced a variety of fragments of Ir by heterologous gene expression in Escherichia coli, and investigated their structures and oligomeric states by circular dichroism (CD) spectroscopy, electron microscopy (EM) and analytical ultracentrifugation (AUC). Our data document that a distinct 14 residue segment exists within Ir that is absolutely required to mediate proper assembly of Ir into a parallel homodimeric coiled coil. Using a 16 residue synthetic peptide (cI-t), we further demonstrate that this coiled-coil 'trigger' site represents an autonomous helical folding unit. These novel findings suggest that coiled-coil formation is controlled by a helical trigger site(s). Results The oligomerization domain of cortexillin I forms a tightly packed, two–stranded, parallel coiled coil Electron microscopic evidence, together with sequence analysis of cortexillin I predicting 18 continuous heptad repeats within its C-terminus, suggested that assembly of the parallel dimeric molecule is mediated by a coiled-coil oligomerization domain (Faix et al., 1996). To test this hypothesis, we produced a recombinant polypeptide chain fragment corresponding to the predicted full-length oligomerization domain comprising residues 227–352 of D.discoideum cortexillin I in E.coli (Figure 1A and B). The homogeneity of the affinity-purified recombinant protein was assessed by tricine-SDS–PAGE (Figure 1C, lane 1) which revealed a single band consistent in size with the calculated molecular mass of the Ir monomer (15.0 kDa), with no obvious degradation products detected. Figure 1.Design and preparation of N- and C-terminal deletion constructs from Ir. (A) Amino acid sequence of Ir. Heptad repeats were assigned according to the COILS algorithm (Lupas et al., 1991) and are shown in blocks of seven amino acid residues denoted abcdefg. Residues at heptad positions a and d are indicated in bold. The amino acid trigger site crucial for coiled-coil formation is underlined. (B) Schematic representation of Ir and various fragments thereof. The sizes of the polypeptide chain fragments are denoted on top by numbers of heptad repeats. The trigger site for coiled-coil formation is marked as a black box. (C) Tricine-SDS–PAGE of purified recombinant Ir fragments under reducing conditions. Lanes: 1, Ir; 2, Ir-2hC; 3, Ir-3hC; 4, Ir-4hC; 5, Ir-5hC; 6, Ir-6hC; 7, Ir–12hC; 8, Ir-12hN; 9, Ir-6hN; 10, cI-310; and 11, cI-324. The migration of marker proteins is given on the right in kDa. Download figure Download PowerPoint The oligomeric state of the recombinant Ir molecule was assessed by AUC. As shown in Table I, at 20°C in 5 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl, the sedimentation equilibrium of Ir revealed an average molecular mass of 27.5 kDa, and sedimentation velocity yielded a sedimentation coefficient of 2.1S. These values are consistent with a dimeric rod-shaped conformation of Ir. Table 1. Mean molar ellipticities at 222 nm, thermal melting temperatures at the midpoint of transition, sedimentation coefficients and molecular masses of the cortexillin I tail domain (Ir), and various Ir fragments and an Ir-derived peptide Fragments [Θ]222a (103 deg.cm2/dmol) Tmb (°C) Sedimentation coefficient s20Wc (S) molecular massd (kDa) Ir −35.4 64 2.10 27.3 (15.0) Ir-2hC −30.9 55 1.58 24.7 (13.3) Ir-3hC −30.4 53 1.76 23.1 (12.6) Ir-4hC −27.5 51 1.60 22.9 (11.7) Ir-5hC −26.3 30 1.61 19.7/14.0e (10.9) Ir-6hC −21.2 n.d. 1.30 10.3 (10.1) Ir-12hC −5.7 n.d. n.d. 4.9 (4.98) Ir-12hN −25.3 27 1.20 8.8 (5.24) Ir-6hN −36.7 63 2.17 39.7 (10.2) cI-310 n.d. n.d. 2.4 37.1 (35.4) cI-324 n.d. n.d. 2.9 71.0 (35.4) cI-t −9.2f n.d. n.d. 2.1g (1.92) All fragments were analyzed in 5 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl, except cI-t which was analyzed in 1 mM sodium phosphate buffer (pH 7.4). The corresponding amino acid sequences are shown schematically in Figure 1B. a [Θ]222 measured at 5°C at chain concentrations (monomer) of 35 μM. b Tm determined at chain concentrations (monomer) of 35 μM. c Sedimentation coefficient and apparent molecular masses were determined at 20°C. The sequence-predicted monomer masses are enclosed in parentheses. Chain concentrations (monomer) varied from 0.1–0.5 mg/ml. d Sedimentation coefficient and apparent molecular masses were determined at 20°C. The sequence-predicted monomer masses are enclosed in parentheses. Chain concentrations (monomer) varied from 0.1–0.5 mg/ml. e Ir-5hC yielded a mixture of two predominant molecular species. f Determined at 3°C at a peptide concentration of 30 μM. g Determined at 3°C at two different peptide concentrations (300 and 30 μM) in the presence and absence of 100 mM NaCl. n.d.: not determined Inspection of the Ir molecules by transmission electron microscopy (TEM) after glycerol-spraying and rotary metal-shadowing yielded uniformly appearing, rod-shaped particles (Figure 2). The apparent length of the molecules was measured and put into a histogram (Figure 2, inset). A Gaussian fit of the histogram revealed a mean length of 19.2 ± 2 nm. This value is consistent with the mean length found for the tail domain of the native cortexillin I molecule (Faix et al., 1996) as well as the calculated length of ∼19 nm for a two-stranded, α-helical coiled-coil consisting of 18 continuous heptad repeats (with the assumption that the axial rise per residue corresponds to 1.485 Å; Fraser et al., 1964). Figure 2.Electron microscopic analysis of recombinant cortexillin Ir homodimer rods. Specimens were prepared by glycerol-spraying/rotary metal-shadowing. Scale bar, 100 nm. Inset, histogram with single Gaussian fit representing the distribution of the molecular length of Ir. One hundred and fifty molecules were measured, with the values displayed representing the mean and standard deviation of the histogram. Download figure Download PowerPoint Far-ultraviolet (UV) CD spectroscopy was employed to probe for the secondary structure of Ir. At 5°C and a total chain concentration of 35 μM, the CD spectrum recorded from Ir (Figure 3A) was characteristic for an α-helical coiled-coil structure exhibiting minima at 208 and 222 nm and a [Θ]222:[Θ]208 ratio of >1 (Zhou et al., 1992). Based on a [Θ]222 value of −35 400 deg.cm2/dmol (Table I), a helical content of >90% was estimated by assuming that a value of −37 300 deg.cm2/dmol corresponds to a helicity of 100% for a 130 residue polypeptide chain fragment (Chen et al., 1974). As expected for a non-covalently bonded, two-stranded α-helical coiled coil, the CD signal at 222 nm was concentration-dependent (data not shown). Figure 3.CD measurements of recombinant cortexillin Ir and various deletion constructs. (A, C and E) Spectra recorded under native conditions at 5°C. (A) Ir. (C) Black curve, Ir-12hC; cyan curve, Ir-12hN; green curve, Ir-6hC; and magenta curve, Ir-6hN. (E) Red curve, Ir-2hC; blue curve, Ir–4hC; and black curve, Ir-5hC. For amino acid sequence, refer to Figure 1A and B. (B, D and F) Thermal unfolding profiles monitored by CD following the temperature-induced change of the mean molar residue ellipticity at 222 nm, [Θ]222. The same color code as for the spectra displayed in (A) and (C) was used. The total chain concentration was 35 μM for all seven fragments in 5 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. Download figure Download PowerPoint The stability of the recombinant two-stranded α-helical coiled coil was assessed by thermal unfolding profiles recorded by CD at 222 nm. As shown in Figure 3B, at a total chain concentration of 35 μM, Ir revealed a single sharp melting profile characteristic for a cooperative helix–coil transition with a melting temperature (Tm) of 64°C. The thermal unfolding profile was monophasic and reversible with >95% of the starting signal being recovered upon cooling (data not shown). To assess whether the hydrophobic core of the coiled coil was tightly packed, binding of the fluorescent dye 1-anilino-8-naphthalenesulfonate (ANS) to Ir was tested (Stryer, 1965; Jelesarov and Bosshard, 1996). Native proteins bind ANS weakly unless there is a solvent-accessible nonpolar region. At 20°C, addition of a 1- to 5-fold molar excess of ANS to a 35 μM Ir solution revealed an increase of the total fluorescence signal of <10% (data not shown) indicating no significant binding of the dye to the coiled coil. To determine whether the helix orientation in the isolated two-stranded coiled-coil domain was parallel, as in the case of the native full-length cortexillin I molecule (Faix et al., 1996), we recombinantly synthesized an Ir variant containing the GlyGlyCys sequence at its C-terminus. Non-reducing SDS–PAGE revealed a single ∼30 kDa protein band, demonstrating that the two Ir chains assemble into a parallel dimer (data not shown). Taken together, these results demonstrate that Ir associates into a tightly packed two-stranded, parallel, α-helical coiled coil which behaves as a single cooperative folding unit. This finding confirms the suggestion made by Faix et al. (1996) that the cortexillins represent a fourth class of molecules within the α-actinin/spectrin superfamily whose members are defined by a common F–actin binding motif, but are distinct from each other mainly in the design and architecture of their oligomerization domain (Matsudaira, 1991). A distinct 14 residue site is necessary for coiled–coil formation of Ir Ir was used as a model system to address the question of whether coiled-coil formation starts preferentially at a distinct site. Towards this goal, our strategy was to produce a number of Ir fragments and assess their ability to form coiled-coil structures by AUC and CD. First we produced, by heterologous gene expression in E.coli, four recombinant polypeptide chain fragments comprising the first six and twelve N-terminal (Ir-12hC and Ir-6hC, respectively) and the last six and twelve C-terminal (Ir-12hN and Ir-6hN, respectively) heptad repeats of Ir (Figure 1B and C). As the minimum length required for the formation of stable coiled coils has been reported to be in the range of 21–23 residues (Lumb et al., 1994; Su et al., 1994; Fairman et al., 1995), these recombinant fragments were expected to be long enough to fold into stable coiled-coil structures. The homogeneity of the affinity-purified recombinant polypeptide chain fragments was assessed by tricine-SDS–PAGE, which revealed single bands with mobilities corresponding to their calculated monomer molecular masses (Table I), with no degradation products detected (Figure 1C). Deletion of six (Ir-6hC) or twelve (Ir-12hC) heptad repeats from the C-terminus of Ir completely abolished coiled-coil formation. AUC analysis of both Ir-6hC and Ir-12hC yielded average molecular masses of 10.3 and 4.9 kDa, respectively, which are consistent with the calculated monomer molecular masses (Table I). Surprisingly, Ir-6hC revealed a CD spectrum indicating a substantial amount of helicity at 5°C (Figure 3C, green curve). However, its CD spectrum was characteristic for partial monomer helix formation as evidenced by its insignificant concentration dependence (data not shown), a shift of the minimum from 208 to 205 nm and a [Θ]222:[Θ]205 ratio of <1. Moreover, its corresponding thermal unfolding profile exhibited a very broad, non-cooperative transition (Figure 3D, green curve). Finally, glycerol-sprayed/rotary metal-shadowed Ir-6hC molecule samples produced specimens which appeared rather heterogeneous in the EM in terms of particle size and shape (data not shown). Ir-12hC, on the other hand, revealed a CD spectrum at 5°C typical for proteins in a random coil conformation with a pronounced minimum at 200 nm (Figure 3C, black curve). As expected, this Ir fragment yielded no significant thermal unfolding profile (Figure 3D, black curve). In contrast, deletion of six (Ir-6hN) or twelve (Ir-12hN) heptad repeats from the N-terminal end of Ir did not significantly affect coiled-coil formation (Figure 3C and D, magenta and cyan curves; Table I). Both fragments yielded concentration-dependent CD spectra characteristic for α-helical coiled-coil structures (Figure 3C), and the corresponding thermal unfolding profiles at a chain concentration of 35 μM exhibited cooperative transitions with Tms of 63 and 27°C for Ir-6hN and Ir-12hN, respectively (Figure 3D; Table I). Rather surprisingly, sedimentation equilibrium revealed that the Ir-6hN fragment forms a tetramer (Table I). Electron micrographs of glycerol-sprayed/rotary metal-shadowed Ir-6hN yielded uniformly distributed, elongated particles which appeared shorter but somewhat thicker than corresponding Ir particles (data not shown). An average molecular length of 12.8 ± 1.2 nm was determined for Ir-6hN, which is exactly two-thirds of the full-length Ir molecule. Together with the data obtained by CD, these findings suggest a four-stranded, most likely unstaggered coiled-coil structure of Ir-6hN. Based on these findings, we next focused our attention on the last six C-terminal heptad repeats (Arg311–Arg352) of Ir. To map more precisely the critical site for coiled-coil formation, we prepared chain variants missing two (Ir-2hC), three (Ir-3hC), four (Ir-4hC) or five (Ir-5hC) heptad repeats from the C-terminus of Ir (Figure 1B and C). Deletions of up to four heptad repeats from the C–terminus of Ir retained the ability of the corresponding Ir fragments for coiled-coil formation (Figure 3E and F; Table I). Ir-2hC (red curve), Ir-3hC (data not shown) and Ir-4hC (blue curve) all had features reminiscent of two-stranded coiled-coil structures as revealed by their α-helical CD spectra (all concentration-dependent; data not shown), cooperative thermal unfolding profiles (Tm values of 55, 53 and 51°C, respectively, at a chain concentration of 35 μM; Table I), and AUC sedimentation velocity profiles and sedimentation equilibrium boundaries characteristic for elongated dimeric molecules (Table I). In contrast, whereas Ir-5hC (Figure 3E and F, black curve) still yielded a mixture of monomers and dimers, deletion of more than five heptad repeats from the C–terminus of Ir resulted in the complete loss of homodimer formation as revealed by AUC (see above and Table I). Taken together, deletion mapping identified a distinct 14 residue site, Arg311–Asp324, which was absolutely necessary to trigger the assembly of the oligomerization domain of cortexillin I into a two-stranded, parallel, α-helical coiled coil. To confirm our findings further, we prepared two cortexillin I fragments, cI-310 and cI-324, which were designed so as to contain the N-terminal globular head domain (residues 1–226) together with 12 (cI-310) or 14 (cI-324) heptad repeats of its coiled-coil domain. Remarkably, electron micrographs of glycerol-sprayed/rotary metal-shadowed cI-310 molecules which were missing the critical sequence Arg311–Asp324 yielded uniformly distributed, apparently monomeric globular particles (Figure 4A). The 4–6 nm diameter particles evidently represented the N-terminal actin-binding domain (residues 1–226 of cortexillin I; Faix et al., 1996). In contrast, cI-324, which differs from cI-310 only in the critical 14 residues Arg311–Asp324 revealed dimeric molecules consisting of two globular heads attached to one end of a ∼15 nm long stalk (Figure 4B). With the exception of a ∼4 nm shorter rod, the overall morphology of the dimeric cI-324 particles appeared very similar to that of glycerol-sprayed/rotary metal-shadowed native cortexillin I (Faix et al., 1996). These EM-based findings were confirmed by AUC measurements yielding average molecular masses of 37.1 kDa (calculated monomer mass 35.4 kDa) for cI-310 and 71.0 kDa (calculated monomer mass 37.1 kDa) for cI-324 (Table I). Figure 4.Electron micrographs of glycerol-sprayed/rotary metal-shadowed recombinant (A) cI-310 and (B) cI-324 cortexillin I fragments which were designed so as to contain the N-terminal globular head domain (residues 1–226) and 12 or 14 heptad repeats, respectively, of the coiled-coil oligomerization domain, Ir. Scale bars, 50 nm for low magnification overviews and 25 nm for high magnification gallery. Download figure Download PowerPoint The coiled-coil 'trigger' site represents an autonomous helical folding unit To characterize the coiled-coil trigger site further, we synthesized the 16 residue peptide Ac-ARMELEARLAKTEKDR-NH2 (Ala310–Arg325), denoted cI-t, and analyzed its structure and oligomeric state by CD and AUC. As illustrated in Figure 5A, at 3°C and in 1 mM sodium phosphate, pH 7.4, the far-UV CD spectrum of cI-t was characteristic for partial helix formation with well-defined minima at 222 nm (α-helix n–π* transition) and 203 nm (mixture of α-helix π–π∥* transition and random coil π–π* transition). Helix formation by the peptide was monomolecular as shown by AUC (Table I) and the lack of any significant concentration dependence of [Θ]222 in the range of 20–200 μM (data not shown). Based on the [Θ]222 value of −9200 deg.cm2/dmol, a helical content of ∼30% was estimated by assuming that a value of −30 000 deg.cm2/dmol corresponds to 100% helicity for a 16 residue peptide (Chen et al., 1974).The monomeric helix formed by cI-t unfolds rapidly with increasing temperature as evidenced by a shift in wavelength of the minimum from 203 to 200 nm at higher temperatures and a concomitant decrease of −[Θ]222 (Figure 5A). Figure 5.CD analysis of the 16 residue peptide cI-t. (A) Far-UV CD spectra at 3, 10, 20 and 50°C in 1 mM sodium phosphate buffer, pH 7.4. The increasing temperature led to a shift in the wavelength of the minimum from 203 to 200 nm and a concomitant decrease of −[Θ]222. (B) pH dependence of [Θ]222 at 3°C (open symbols) and at 50°C (filled symbols) in 1 mM sodium citrate, 1 mM sodium phosphate and 1 mM sodium borate buffer. (C) NaCl dependence of [Θ]222 at 3°C in 1 mM sodium phosphate buffer, pH 7.4. The peptide concentration was 30 μM for all experiments. Download figure Download PowerPoint The helix content of cI-t has also been measured as a function of pH and ionic strength. The helix content of cI-t at 3°C was strongly pH-dependent: −[Θ]222 decreased moderately as the pH was raised above 2 and decreased significantly as the pH was increased above 11 (Figure 5B, open symbols). This pH effect was drastically attenuated at 50°C where the helix was largely melted (Figure 5B, filled symbols). A strong decrease in the helix content of cI-t was also observed upon raising the ionic strength from 0 to 2 M NaCl (Figure 5C). Throughout the pH range and salt concentrations evaluated, helix formation remained temperature-dependent and occurred in a monomolecular reaction. These results suggest that electrostatic interactions of charged amino acid side chains are critical in stabilizing the cI-t monomer helix. Discussion A coiled-coil trigger site is absolutely necessary for chain assembly of cortexillin I The two-stranded parallel coiled coil is the simplest representative of multi-subunit proteins with only one type of secondary- and a well-defined quartenary-structure. This structural motif has been used extensively as a model system for studying both the intra- and intermolecular interactions which govern the folding and stability of multimeric proteins. As a consequence of (i) thermodynamic studies of naturally occurring coiled-coil proteins (Lehrer, 1978; O'Shea et al., 1989), (ii) the design an
Referência(s)