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Critical interactions in the stability control region of tropomyosin.

ABSTRACT: Our laboratory has recently described a stability control region in the two-stranded alpha-helical coiled-coil alpha-tropomyosin that accounts for overall protein stability but is not required for folding (Hodges et al., 2009). We have used a synthetic peptide approach to investigate three stability control sites within the stability control region (residues 97-118). Two of the sites, electrostatic cluster 1 (97-104, EELDRAQE) and electrostatic cluster 2 (112-118, KLEEAEK), feature sequences with unusually high charge density and the potential to form multiple intrachain and interchain salt bridges (ionic attractions). A third site (105-111, RLATALQ) features an e position Leu residue, an arrangement known previously to enhance coiled-coil stability modestly. A native peptide and seven peptide analogs of the tropomyosin sequence 85-119 were prepared by Fmoc solid-phase peptide synthesis. Thermal stability measurements by circular dichroism (CD) spectroscopy revealed the following T(m) values for the native peptide and three key analogs: 52.9 degrees C (Native), 46.0 degrees C (R101A), 45.3 degrees C (K112A/K118A), and 27.9 degrees C (L110A). The corresponding DeltaT(m) values for the analogs, relative to the native peptide, are -6.9 degrees C, -7.6 degrees C, and -25.0 degrees C, respectively. The dramatic contribution to stability made by L110e is three times greater than the contribution of either electrostatic cluster 1 or 2, likely resulting from a novel hydrophobic interaction not previously observed. These thermal stability results were corroborated by temperature profiling analyses using reversed-phase high-performance liquid chromatography (RP-HPLC). We believe that the combined contributions of the interactions within the three stability control sites are responsible for the effect of the stability control region in tropomyosin, with the Leu110e contribution being most critical.

SUBMITTER: Kirwan JP

PROVIDER: S-EPMC2856757 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Critical interactions in the stability control region of tropomyosin.

Kirwan J Paul JP Hodges Robert S RS

Journal of structural biology 20100206 2

Our laboratory has recently described a stability control region in the two-stranded alpha-helical coiled-coil alpha-tropomyosin that accounts for overall protein stability but is not required for folding (Hodges et al., 2009). We have used a synthetic peptide approach to investigate three stability control sites within the stability control region (residues 97-118). Two of the sites, electrostatic cluster 1 (97-104, EELDRAQE) and electrostatic cluster 2 (112-118, KLEEAEK), feature sequences wit ...[more]

PMID: 20144718

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Project description:Experimental approaches such as fiber diffraction and cryo-electron microscopy reconstruction have defined regulatory positions of tropomyosin on actin but have not, as yet, succeeded at determining key atomic-level contacts between these proteins or fully substantiated the dynamics of their interactions at a structural level. To overcome this deficiency, we have previously employed computational approaches to deduce global dynamics of thin filament components by energy landscape determination and molecular dynamics simulations. Still, these approaches remain computationally challenging for any complex and large macromolecular assembly like the thin filament. For example, tropomyosin cable wrapping around actin of thin filaments features both head-to-tail polymeric interactions and local twisting, both of which depart from strict superhelical symmetry. This produces a complex energy surface that is difficult to model and thus to evaluate globally. Therefore, at this stage of our understanding, assessing global molecular dynamics can prove to be inherently impractical. As an alternative, we adopted a "divide and conquer" protocol to investigate actin-tropomyosin interactions at an atomistic level. Here, we first employed unbiased protein-protein docking tools to identify binding specificity of individual tropomyosin pseudorepeat segments over the actin surface. Accordingly, tropomyosin "ligand" segments were rotated and translated over potential "target" binding sites on F-actin where the corresponding interaction energetics of billions of conformational poses were ranked by the programs PIPER and ClusPro. These data were used to assess favorable interactions and then to rebuild models of seamless and continuous tropomyosin cables over the F-actin substrate, which were optimized further by flexible fitting routines and molecular dynamics. The models generated azimuthally distinct regulatory positions for tropomyosin cables along thin filaments on actin dominated by stereo-specific head-to-tail overlap linkage. The outcomes are in good agreement with current cryo-electron microscopy topology and consistent with long-thought residue-to-residue interactions between actin and tropomyosin.

Project description:BackgroundNemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of nebulin is composed of approximately 35-residue α-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site.MethodsWe produced four wild-type (WT) nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT nebulin super repeats for WT α- and β-tropomyosin, and for β-tropomyosin with six patient mutations: p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro.ResultsWT nebulin was shown to interact with actin and tropomyosin. Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p.Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18).ConclusionsWe demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.

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Critical interactions in the stability control region of tropomyosin.

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Critical interactions in the stability control region of tropomyosin.

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