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Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635
Correspondence: Address reprint requests to Norma J. Greenfield, Tel.: 732-235-5791; Fax: 732-235-4029; E-mail: greenfie{at}rwja.umdnj.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-TM. Mutation of L27 to E or G in the Tmod helix reduces TM affinity. Residues 49–92 are required for capping but do not bind TM. Of these, residues 67–75 have the sequence of an amphipathic helix, but are not helical. Residues 55–62 and 76–92 display negative 1H-15N heteronuclear Overhauser enhancements showing they are flexible. The conformational dynamics of these residues may be important for actin capping activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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; Pollard and Borisy, 2003). The actin filaments found in the sarcomeres of striated muscles, as well as in stress fibers and microvilli in nonmuscle cells, are stable, unbranched structures. Actin binding proteins that regulate addition and loss of actin subunits at the two ends of the filaments, and stabilize the filament against severing proteins, are needed to maintain stable actin filaments. A class of proteins referred to as capping proteins block the rapidly growing, "barbed" or "plus" end (Cooper and Schafer, 2000). In addition, two proteins, tropomyosin (TM) and tropomodulin (Tmod), act together to regulate the dynamics of the slow growing, "pointed", or "minus" end of the filament (Fowler, 1990, 1997; Fowler et al., 2003; Kostyukova and Hitchcock-DeGregori, 2004; Weber et al., 1999).
The tropomodulins, which cap the pointed end of the actin filaments, are a family of widely expressed proteins that bind both to TM and actin (reviewed in Conley, 2001; Conley et al., 1998; Fowler, 1997; Fowler and Conley, 1999). Tropomodulin 1 (Tmod1, E-Tmod) was originally isolated from human erythrocytes as a tropomyosin-binding protein (Fowler, 1987, 1990), and is also the prevalent form in mammalian cardiac muscle and slow skeletal muscle. Other isoforms include Tmod2, found only in neuronal tissue (Cox and Zoghbi, 2000; Sussman et al., 1994; Watakabe et al., 1996), Tmod3 (ubiquitous Tmod), which is widely expressed in a variety of different organs and tissues (Cox and Zoghbi, 2000), and Tmod4 (Sk-Tmod), restricted to skeletal muscle (Almenar-Queralt et al., 1999; Conley et al., 2001; Cox and Zoghbi, 2000). Tropomodulin has been localized to the pointed end of actin filaments in the sarcomeres of striated muscle (Fowler et al., 1993), in the spectrin-actin cytoskeleton in erythrocytes (Ursitti and Fowler, 1994) and in cultured cells (Fischer et al., 2000, 2003a).
The most extensive functional analysis has been of Tmod1 using native and recombinant protein, and protein fragments, although Tmod3 and Tmod4 have also been shown to block actin filament elongation in vitro (Fischer et al., 2003a,b). Full-length Tmod1 can bind TM and block actin filament polymerization and depolymerization in a TM-dependent fashion (Fowler, 1990; Weber et al., 1994, 1999). Tropomodulin 1 has two domains, which are functionally and structurally distinct (Fujisawa et al., 2001; Kostyukova et al., 2000, 2001). The C-terminal half is the major actin binding domain and at high concentrations can cap the pointed end of filaments in the absence of TM. In addition, it weakly nucleates polymerization (Fowler et al., 2003). This domain also binds nebulin (Krieger et al., 2002; McElhinny et al., 2001). The domain unfolds in a two-state transition and its atomic structure is a right-handed superhelix composed of alternating -helices and ß-strands (Krieger et al., 2002). The C-terminal folded region contains a leucine-rich repeat domain, which is involved in many protein-protein interactions (Kobe and Deisenhofer, 1994).
The N-terminal domain of Tmod1 binds TM in an isoform-specific manner (Babcock and Fowler, 1994; Greenfield and Fowler, 2002; Kostyukova and Hitchcock-DeGregori, 2004; Sung et al., 1992; Sussman and Fowler, 1992) and it becomes more structured upon binding to TM (Greenfield and Fowler, 2002; Kostyukova and Hitchcock-DeGregori, 2004; Kostyukova et al., 2001). The structure of the N-terminal region of Tmod1 is elongated and disordered (Fujisawa et al., 2001; Kostyukova et al., 2001), and attempts to crystallize it and full-length Tmod1 have failed. It can cap actin filaments in the presence of TM (Fowler et al., 2003; Kostyukova and Hitchcock-DeGregori, 2004). The interaction between the N-terminal domain of Tmod1 and TM is required to maintain stable thin filaments in cardiac myocyte sarcomeres (Mudry et al., 2003).
In this work we used high-resolution NMR and circular dichroism (CD) to characterize the structure of the N-terminal capping domain of Tmod and to define precisely the TM binding region. In this study we used three fragments of Tmod containing residues 1–92 (Tmod11-92), 1–48 (Tmod11-48) (Kostyukova and Hitchcock-DeGregori, 2004), and 38–92 (Tmod138-92). In the presence of TM, Tmod11-92, inhibits both polymerization and depolymerization at the actin filament's pointed end. Tmod11-48 retains full affinity for TM, but it has no capping activity (Kostyukova and Hitchcock-DeGregori, 2004). Our results show that Tmod11-92 has two domains that differ in structure and flexibility. The N-terminal domain is partially ordered and contains the TM binding site, whereas the C-terminal end, necessary for capping activity, is extended and flexible. Mutagenesis of a hydrophobic residue in the observed short helical region (residues 24–35) shows that a coiled-coil motif is required for high affinity TM binding, suggesting that part of the TM/Tmod1 interface contains a triple helical region.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). The peptides contain a His tag (a Met residue followed by six histidines) at the N-terminus. To prepare Tmod11-92 labeled with 15N alone or 15N and 13C, the proteins were expressed in minimal medium containing 15N ammonium sulfate with or without glucose uniformly labeled with 13C as previously described (Greenfield et al., 2001). Tmod138-92 was synthesized and purified by the Keck Facility at Yale University (New Haven, CT). Unlabeled TM1a1-14Zip (also called GlyTM1aZip, Palm et al., 2003; or TM1a1-14, Greenfield et al., 2002a) and TM1b1-19Zip (also called GlyTM1bZip; Greenfield and Fowler, 2002), chimeric peptides containing the first 14 residues of the N-terminus of rat striated muscle TM encoded by exon 1a or the first 19 residues of the N-terminus of rat nonmuscle TMs encoded by exon 1b, respectively (Helfman et al., 1986), and the last 18 C-terminal residues of the yeast transcription factor GCN4 (Landschulz et al., 1988) and with glycines substituted for the N-terminal acetyl groups, were prepared as previously described (Greenfield et al., 2001, 2002b). Purified acetylated TM1b1-19Zip (AcTM1b1-19Zip) and TM1a1-14Zip (AcTM1a1-14Zip), which have an acetyl group instead of glycine at the N-terminus, were purchased from SynPep (Dublin, CA). Chicken muscle -tropomyosin was prepared as previously described (Hitchcock-DeGregori et al., 1985).
Preparation of Tmod11-92L27E and Tmod11-92L27G
Mutations were introduced into DNA encoding Tmod11-92 using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmids were amplified by PCR using PfuTurbo DNA polymerase with the plasmid pET(His)Tmod11-92 (Kostyukova et al., 2004) as the template using two complementary sets of oligonucleotides. To change L27 to G the oligonucleotides were:
To change L27 to E the oligonucleotides were:
The mutations are underlined. The original plasmid was digested using Dpn I and the mixture was used to transform Escherichia coli (DH5). The presence of the mutations was confirmed by sequencing of the full tropomodulin insert. Synthesis of all oligonucleotides and sequence determination were done at the University of Medicine and Dentistry of New Jersey DNA synthesis and sequencing facility (Robert Wood Johnson Medical School, Piscataway, NJ).
The mutated Tmod11-92 fragments were overexpressed in E. coli strain BL21(DE3)pLysE and purified as described for the wild-type Tmod11-92 fragment (Kostyukova and Hitchcock-DeGregori, 2004). The molecular masses of these fragments, determined by electrospray mass spectroscopy (Keck Biotechnology Resource Laboratory, Yale University), were 11,820 found versus 11,820.1 predicted for L27E and 11,748 found versus 11,748.1 predicted for L27G. Their purity was determined using SDS-PAGE (Laemmli, 1970).
Electrophoresis
Native gel electrophoresis of the Tmod fragments and mutants and their 1:1 mixtures with AcTM1b1-19Zip were performed in 9% polyacrylamide gels that were polymerized in the presence of 10% glycerol without SDS at pH 8.8 as previously described (Greenfield et al., 2002b). To prepare the complexes for loading on the gel, stock solutions containing 48 µM Tmod peptide in the absence or presence of 48 µM AcTM1b1-19Zip in 100 mM NaCl, 10 mM sodium phosphate, pH 6.5, were mixed 1:1 with buffer containing 20 mM Tris, pH 8.8, 20% glycerol, plus 0.004% bromphenol blue as a marker. Fifteen microliters of each sample were loaded on the gels.
Determination of protein concentration
The concentrations of Tmod11-48, the wild-type and mutated Tmod11-92 peptides, the TM1a1-14Zip and TM1b1-19Zip peptides, and chicken muscle -tropomyosin were determined by measuring the difference spectra of their tyrosine residues in 6 M guanidine-HCl between pH 12.5 and 6.0 (Edelhoch, 1967) using an extinction coefficient of 2357 per tyrosine residue (Fasman, 1989). The concentration of Tmod138-92 was estimated from its phenylalanine absorption at 257.6 nm in 100 mM NaCl, 10 mM sodium phosphate, pH 6.5, using a molar extinction coefficient of 195.1 per phenylalanine (Fasman, 1989).
Circular dichroism spectroscopy, secondary structure estimation, and thermodynamics of folding
CD measurements were carried out on Aviv Model 62D (Aviv Biomedical, Lakewood, NJ) or Aviv Model 215 (Proterion, Piscataway, NJ) spectrophotometers. All measurements were made in 100 mM NaCl, 10 mM sodium phosphate, pH 6.5. Protein and peptide concentrations ranged from 5 to 20 µM. The secondary structures of Tmod11-92 and Tmod11-48 were estimated from the mean residue ellipticity between 260 and 200 nm using three computer programs: CDNN, a neural net analysis method that uses a protein reference set (Bohm et al., 1992); SELCON1, a self-consistent singular value decomposition method with a protein database as standards (Sreerama and Woody, 1994), and LINCOMB, a constrained least squares analysis method (Perczel et al., 1992), using a fixed peptide reference set for -helix, ß-structure, ß-turn, and random coil (Brahms and Brahms, 1980). The enthalpies and TMs of folding of the Tmod1 peptide/TM peptide complexes were measured by following the ellipticity at 222 nm as a function of temperature as previously described (Greenfield and Fowler, 2002).
NMR spectroscopy of Tmod11-92
NMR data of unbound Tmod11-92 were collected on a Varian Inova 500 spectrometer (Varian, Palo Alto, CA) at 10°C. The concentration of 15N labeled peptide was 1.1 mM and that of the 13C15N labeled peptide was 0.8 mM. Measurements of the complex of 13C15N-Tmod1-92, 0.9 mM with an excess of unlabeled TM1a1-14Zip, 1.3 mM, were made on a Varian Inova 600 spectrometer (Varian) at 10 and 20°C. All spectral measurements were made in susceptibility-matched NMR tubes (Shigemi, Allison Park, PA) in 100 mM NaCl, 10 mM sodium phosphate, 5% D2O, pH 6.5. The programs VNMR (Varian) and NMRpipe (Delaglio et al., 1995) were used for data processing. All of the spectra were processed with twofold zero filling, Gaussian or sine bell multiplication, and linear prediction. The program Sparky (T. Goddard and T. Kneller, University of California at San Francisco, unpublished) was used for peak peaking and determining the intensity of the crosspeaks.
Resonance assignments
The assignments of the backbone resonances of the N, H, C Cß, C', and H atoms were determined from spectra acquired using NMR pulse sequences that transfer magnetization through bonds including 15N-1H-heteronuclear single quantum correlation (HSQC) (Bodenhausen and Ruben, 1980; Kay et al., 1992), H(CA)NH and CANH (Feng et al., 1996; Montelione and Wagner, 1990), H(CA)(CO)NH and CA(CO)NH (Boucher et al., 1991; Feng et al., 1996), and CBCANH (Grzesiek and Bax, 1992a,b; Rios et al., 1996), as previously described (Greenfield et al., 2003). In addition, an HNN experiment (Panchal et al., 2001) was performed to confirm some of the assignments of the 15N and 1H backbone resonances. Side-chain resonance assignments were made using two three-dimensional (3D) correlated spectroscopy (cosy) pulse sequences, HCCHcosy and CCHcosy (Bax et al., 1990; Kay et al., 1990) and a 1H-13C-HSQC (Bodenhausen and Ruben, 1980) spectrum. Two-dimensional planes of 13C edited nuclear Overhauser enhancement and exchange spectroscopy (NOESY) spectra (Ikura et al., 1990; Pascal et al., 1994) of the aromatic region were used to assign the side-chain resonances of the aromatic resonances of the tyrosines. Most of the aromatic resonances arising from the histidine and phenylalanine resonances were not resolved and could not be assigned.
Determination of the structure and dynamic properties of Tmod11-92
Distance constraints for structure analysis of the uncomplexed Tmod11-92 fragment were obtained by analysis of 3D 15N edited pulsed field gradient (PFG) NOESY-HSQC (Driscoll et al., 1990; Pascal et al., 1994) and 13C edited PFG NOESY-HSQC (Ikura et al., 1990; Pascal et al., 1994) experiments, with mixing times of 80 and 100 ms, respectively. The intensities were converted to distances and the structure was calculated using AutoStructure v1.2.1 (Huang et al., 2003; Moseley and Montelione, 1999; Zheng et al., 2003) as previously described (Greenfield et al., 2001, 2003). Heteronuclear 15N-1H Overhauser enhancement (HNOE) data were collected on the Tmod11-92/TM1a1-14Zip peptide complex to characterize the relative flexibility of the Tmod residues when bound to the TM peptide (Kay et al., 1989; Li and Montelione, 1994a, 1995).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). A fragment of Tmod1 that contains residues 1–92, originally identified in preparations of full-length Tmod1 expressed in E. coli (Kostyukova et al., 2000) (Fig. 1), retains these functions (Kostyukova et al., 2000; Kostyukova and Hitchcock-DeGregori, 2004). Here we determined the structure of this fragment, identified the TM binding site within it, and defined the structural requirements for TM binding using site-directed mutagenesis.
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). The percent helical contents, estimated using three different computer programs (see Materials and Methods), were 24 ± 5, 22 ± 1, 15 ± 2, and 20 ± 2, respectively. The agreement among the programs for the estimates of other secondary structures was poor, showing the only regular secondary structural element is -helix and that the N-terminal half of Tmod1-92 has a higher helical content than the C-terminal half.
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and 13C chemical shift displacements (Fig. 4 A) and the pattern of 
to 
to 
to 
and 
to 
nuclear Overhauser enhancements (NOEs) (Fig. 4 B) show that residues 24–35 are -helical (Wang and Jardetzky, 2002; Wishart and Sykes, 1994; Wishart et al., 1992). The presence of Hi to 
NOEs suggests there may be some poorly defined regions with turns between residues 18–23 and 61–70 (Wüthrich, 1986). The minor displacements of the rest of the resonances indicate there is little other regular secondary structure.
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; Cuff et al., 2000). The discrepancy arises because optical transitions, which occur on the order of femtoseconds, can detect secondary structures that are present for short time periods, whereas NOEs characteristic of secondary structures take much longer to develop (on the order of milliseconds). For example, residues 65–75 have the sequence of an amphipathic helix, and although they do not form a stable structure, they may contribute to the helical content measured using CD. There are often discrepancies between the helical content measured by CD and by characteristic NOEs in NMR spectra in partially folded proteins with the characteristics of molten globules. For example, in a recent study of the lactalbumin molten globule (Ramboarina and Redfield, 2003) the protein was highly helical when measured by CD but no NOEs characteristic of -helices were observed.
Mapping the TM binding site in Tmod1
NMR experiments localized the binding site for TM within residues 1–35 of Tmod1. When complexed with TM1a1-14Zip, a peptide containing the N-terminus of long TMs found in muscle and nonmuscle cells, the crosspeaks in the 1H-15N-HSQC spectra originating from residues 1–35 of Tmod11-92 were completely broadened and/or shifted at 10°C and only peaks from residues 36–92 (Fig. 3, black crosspeaks) were observed. Only a few low-intensity shifted crosspeaks originating from the 35 N-terminal residues were observed. Their HN resonances could not be assigned because there were almost no crosspeaks to them in the set of through-bond spectra used for sequential assignments. The crosspeaks from residues 36–47 were slightly shifted and broadened, but there was no significant effect on the crosspeaks of residues 48–92. Thus, the direct binding site lies within residues 1–35, although TM binding may influence the conformation of residues 36–47.
The NMR spectra of the complex were initially obtained at 10°C to maximize the structure of the Tmod fragments. To determine if the broadening of the NMR spectra at 10°C was due to slow tumbling resulting from formation of a rigid rod upon binding TM1a1-14, the temperature of the Tmod/TM complex was raised to 20°C to increase the rate of tumbling. At 20°C, 
30 new well-dispersed peaks appeared in the 1H-15N HSQC spectrum (Fig. 6) arising from residues within the TM binding region. Only the crosspeaks from residues 20–26 could be unambiguously assigned due to low signal/noise in most of the 3D NMR spectra needed for resonance assignment and structure determination. The chemical shifts of the C and H resonances of residues 20–26 suggest that residues 20–23 do not become helical, but 24–26 remain helical upon complex formation. Residues 20–26 have positive 15N-1H HNOEs (Fig. 6, magenta crosspeaks) showing that they are ordered in the complex. In addition, many of the new unassigned crosspeaks in the HSQC spectrum are associated with positive HNOEs showing that most of the TM binding region becomes ordered upon complex formation. The resonances arising from residues 40–92, outside of the TM binding region, are only slightly displaced by raising the temperature from 10 to 20°C. The results show that the low signal/noise of the resonances in the TM binding site at 10°C is probably due to slow tumbling rather than broadening from conformational exchange of Tmod1 residues within the binding site. CBCACONH and CBCANH spectra were obtained at 750 MHz in addition to those obtained at 500 MHz, but the higher field strength did not improve the signal/noise.
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-tropomyosin (-TM). As expected from the NMR binding results, the Tmod138-92 peptide does not bind to AcTM1b1-19Zip as measured using CD (Fig. 7 C) or native gel electrophoresis (results not shown). Tmod138-92 also has no effect on the stability of full-length chicken muscle striated -TM (data not shown), in contrast to the effect of Tmod11-92, which increases the stability and cooperativity of folding of TM (Kostyukova et al., 2001).
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-helix (residues 24–35) within the TM binding site is in a region with coiled coil propensity (Fig. 1, residues 23–38) (Lupas et al., 1991), but the lack of periodicity of the displacements of the 1HN resonances in the helical region showed that Tmod11-92 does not contain a coiled coil (Goodman and Kim, 1991). Because the CD studies showed that the helical content of Tmod11-92 increases when it binds to TM, we designed mutants to test the importance of the amphipathic coiled-coil motif for binding to TM. Leu-27 at a hydrophobic interface a position in a heptapeptide repeat was mutated to Gly, which would destabilize the -helix (O'Neil and DeGrado, 1990), or Glu, whose charged side chain would destroy a hydrophobic coiled-coil interaction, without perturbing the -helix. We tested binding to AcTM1b1-19Zip using native gel electrophoresis and CD (Fig. 7). Neither mutant formed a stable complex with AcTM1b1-19Zip suggesting that part of the Tmod/TM interface forms a three-chained coiled coil.
The specificity of Tmod1-TM binding is within Tmod1 residues 1–48
Studies with full-length TMs and model peptides containing the N-termini of long and short TMs, TM1a1-14Zip, and TM1b1-19Zip (Greenfield and Fowler, 2002) show that Tmod1, as well as the N-terminal fragments, Tmod11-130 and Tmod11-92, have higher affinity for short, 247-residue than long, 284-residue tropomyosins (Babcock and Fowler, 1994; Greenfield and Fowler, 2002; Kostyukova and Hitchcock-DeGregori, 2004). It was possible that the higher affinity of the Tmod11-92 peptide for the 1b peptide is influenced by the C-terminal region of the Tmod fragment, even though the C-terminal peptide, Tmod138-92 does not bind to TM. Here we show that the binding specificity resides within residues 1–48. Fig. 8 shows the temperature-induced unfolding of AcTM1a1-14Zip and AcTM1b1-19Zip in the absence or presence of Tmod11-48. In the absence of the Tmod fragment the two TM peptides had similar enthalpies of unfolding, 31.5 and 31.9 Kcal/mol, respectively. The unfolding and refolding of the TMZip peptides and the Tmod1-92/TMZip complexes were completely reversible. Mixing with Tmod11-48 increased the TMs of unfolding by 12.0 and 12.6°C, respectively. The enthalpies of unfolding of the complexes were 47.7 and 73.8 Kcal/mol, respectively. The higher TM and enthalpy of unfolding of the 1b complex than the 1a complex reflect tighter binding (reviewed in Greenfield, 2004a,b) showing that the binding specificities for the tropomyosin isoforms is a property of residues 1–48.
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) of Tmod1 and of long TMs to residues 6–94 (Babcock and Fowler, 1994). Although we cannot fully explain the discrepancies, the earlier studies measured binding of 125I-TM to immobilized Tmod on a nitrocellulose membrane. In our hands, 125I-TM was not an optimal ligand. Degradation of TM was evident within days of labeling with 125I and after six weeks no full-length 125I-TM remained when examined by gel electrophoresis. Here, and in previous studies from our laboratories, three independent, direct methods (native gel electrophoresis, CD, and NMR) have given consistent results. Most relevant, the broadening of NMR spectra is a well-established method for showing that residues are in a binding site, especially when broadening is confined to specific regions of the molecule, as it is here.
The flexibility of Tmod11-92
Analysis of the "through bond" NMR spectra used to assign the backbone resonances (see Materials and Methods) showed that the N- and C-terminal halves of Tmod11-92 have different dynamics. In this class of spectra crosspeaks may be broadened by transverse relaxation (also called spin-spin or T2 relaxation) if a protein or a region of a protein has a large radius of gyration and tumbles slowly, or by exchange broadening if residues switch between multiple conformations. Proline-39, conserved in all Tmods and related proteins (Fig. 1), defines a boundary between two domains in Tmod11-92: the TM binding site and the region required for TM-dependent capping of the actin filament. The residues preceding P39 contain the only helical region, residues 24–35, and most of the residues with interresidue NOEs (Fig. 4 B). Residues in this region have through-bond crosspeaks with relatively low intensities (Fig. 9). In contrast, most of the crosspeaks to H, C, and Cß resonances originating from residues 40–92 in the through-bond spectra are sharp and 3–4 times more intense than those from residues preceding P39, suggesting that the C-terminal region is more flexible and disordered than the N-terminal end of Tmod11-92.
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-and ß-carbon atom sequential peaks from P58 to the T59 HN and P61 to the F62 HN indicate that the higher intensity peaks originate from residues following trans prolines. The preferred trans proline state favors an elongated conformation, consistent with the x-ray scattering results (Fujisawa et al., 2001).
The spectra of the Tmod11-92/TM1a1-14Zip complex at 20°C give additional information about the structure and dynamics of the C-terminal domain of the Tmod1 peptide. Fig. 6 shows the 15N-1H HSQC spectrum overlaid with a heteronuclear 15N-1H HNOE spectrum (Li and Montelione, 1994b). The crosspeaks of residues 40–92 from Tmod in the complex had high intensities in the through-bond spectra that were used for sequential assignments, and the HN resonances in this region were all assigned. Residues 40–54 have positive 1H-15N-HNOEs showing they are ordered in the complex whereas residues 40–48 are disordered in the unbound Tmod11-92, indicating they become more ordered when TM binds. Residues 65–72 are also ordered, based on their positive HNOEs. These residues, which are not affected by TM1a1-14Zip binding, have lower than average intensities in the through-bond spectra of the uncomplexed peptide (e.g., see Fig. 9) suggesting there may be conformational exchange of these residues between ordered and less-ordered conformations. Residues 79–92, nearly identical in the vertebrate Tmods, are flexible, displaying negative HNOEs.
Relationship of the structure to tropomodulin family sequence homologies
To consider whether the structural domains and TM binding site we have identified within Tmod11-92 are conserved in other members of the tropomodulin family, we compared their sequences to the first 92 residues of chicken Tmod1 in Fig. 1. We aligned the sequences encoded by representatives of four Tmod genes (Tmod1–Tmod4) found throughout vertebrates, three invertebrate homologs, and three mammalian leiomodin (Lmod) genes whose functions are unknown (Conley, 2001; Conley et al., 2001).
Although the extreme N-termini are variable, all of the Tmod sequences can be aligned with chicken Tmod1 starting at residue L7 and with the Lmods starting at residue D14. The pattern of hydrophobic and hydrophilic residues in the Tmods, corresponding to residues 7–34 in chicken Tmod1, is almost completely conserved suggesting that they have similar folds. This pattern is also substantially conserved in the Lmods. There are, however, significant sequence differences among vertebrate Tmod isoforms that may be responsible for the affinity differences of Tmod isoforms for TM isoforms (Greenfield and Fowler, 2002).
The region containing residues 39–70, following the TM binding domain, is the most conserved among Tmods and Lmods: P39, P45, K56, G60, and R64 are identical throughout and another six residues are highly homologous (same charge and hydrophobicity). Residues 55–62, containing the conserved T59 and G60, are flexible as evidenced by the negative HNOEs of the residues in this region and the cis/trans isomerization of P58 and P61. The conformational dynamics of this conserved region may be important for binding to actin with TM. Residues 71–92 are highly conserved among the vertebrate Tmods (17 conserved residues), but less well conserved with the invertebrate sequences (seven conserved residues) and not conserved with the Lmods. Residues 67–75 in the Tmods have an amphipathic helix-like sequence, often implicated in protein-protein interactions. This region is not helical in Tmod11-92 in the presence or absence of TM, although it may become helical when Tmod is bound to actin.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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This work was funded by a Grant-In-Aid from the American Heart Association Heritage Affiliate to N.J.G. (0256468T), and by grants from the National Institutes of Health to S.E.H-D. (GM63257) and to S.E.H-D. and N.J.G. (GM36326).
Submitted on August 6, 2004; accepted for publication September 27, 2004.
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) Tmod11-130, 10 µM, (•) Tmod11-92, 1 10 µM, (
) Tmod11-48, 10 µM, and (
)Tmod138-92, 20 µM. All peptides were in 100 mM NaCl, 10 mM sodium phosphate, pH 6.5, 0°C.
