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A peek into tropomyosin binding and unfolding on the actin filament.

Singh A, Hitchcock-Degregori SE - PLoS ONE (2009)

Bottom Line: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding.The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample.We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America. Abhishek.Singh@ucsf.edu

ABSTRACT

Background: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin.

Principal findings: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism).

Conclusions: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

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Related in: MedlinePlus

Models illustrating the P2 and P3 regions in wildtype and mutant tropomyosins.The side chains of the alanine clusters (magenta) and consensus actin binding sites (cyan) [13] are illustrated on a ribbon model of the 7 Å structure (pdb ID: 1C1G; [60]). Period 2, residues 46–69, and period 3, residues 88–111, are enlarged with the side chains of interface Ala residues (in magenta, space filling), canonical interface residues (green) and consensus residues (cyan, Cβ in spacefill). The colors used are the same as in Table 1.
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pone-0006336-g001: Models illustrating the P2 and P3 regions in wildtype and mutant tropomyosins.The side chains of the alanine clusters (magenta) and consensus actin binding sites (cyan) [13] are illustrated on a ribbon model of the 7 Å structure (pdb ID: 1C1G; [60]). Period 2, residues 46–69, and period 3, residues 88–111, are enlarged with the side chains of interface Ala residues (in magenta, space filling), canonical interface residues (green) and consensus residues (cyan, Cβ in spacefill). The colors used are the same as in Table 1.

Mentions: We designed mutants to transform secondary sites (P2, P3) into primary sites by introducing an Ala cluster in place of the native, more stable interface. To do this an Ala was “shifted” into the consensus region to mimic the Ala clusters at the P1 and P5 interfaces, creating the P2Shift and P3Shift mutants (Figure 1, Table 1; P2Shift = Y60A/L64A; P3Shift = L106A/A120L). In P3Shift we introduced A120L mutation C-terminal to P3 as a compensatory mutation since P3 is adjacent to the least stable region of the molecule (residues 130–190). In addition, we created P2P3Shift, combining the above mutations to create four primary sites. Based on studies with model peptides, the Ala substitutions in the context of P2 and P3 would destabilize the coiled coil [16], [21].


A peek into tropomyosin binding and unfolding on the actin filament.

Singh A, Hitchcock-Degregori SE - PLoS ONE (2009)

Models illustrating the P2 and P3 regions in wildtype and mutant tropomyosins.The side chains of the alanine clusters (magenta) and consensus actin binding sites (cyan) [13] are illustrated on a ribbon model of the 7 Å structure (pdb ID: 1C1G; [60]). Period 2, residues 46–69, and period 3, residues 88–111, are enlarged with the side chains of interface Ala residues (in magenta, space filling), canonical interface residues (green) and consensus residues (cyan, Cβ in spacefill). The colors used are the same as in Table 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2710508&req=5

pone-0006336-g001: Models illustrating the P2 and P3 regions in wildtype and mutant tropomyosins.The side chains of the alanine clusters (magenta) and consensus actin binding sites (cyan) [13] are illustrated on a ribbon model of the 7 Å structure (pdb ID: 1C1G; [60]). Period 2, residues 46–69, and period 3, residues 88–111, are enlarged with the side chains of interface Ala residues (in magenta, space filling), canonical interface residues (green) and consensus residues (cyan, Cβ in spacefill). The colors used are the same as in Table 1.
Mentions: We designed mutants to transform secondary sites (P2, P3) into primary sites by introducing an Ala cluster in place of the native, more stable interface. To do this an Ala was “shifted” into the consensus region to mimic the Ala clusters at the P1 and P5 interfaces, creating the P2Shift and P3Shift mutants (Figure 1, Table 1; P2Shift = Y60A/L64A; P3Shift = L106A/A120L). In P3Shift we introduced A120L mutation C-terminal to P3 as a compensatory mutation since P3 is adjacent to the least stable region of the molecule (residues 130–190). In addition, we created P2P3Shift, combining the above mutations to create four primary sites. Based on studies with model peptides, the Ala substitutions in the context of P2 and P3 would destabilize the coiled coil [16], [21].

Bottom Line: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding.The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample.We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America. Abhishek.Singh@ucsf.edu

ABSTRACT

Background: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin.

Principal findings: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism).

Conclusions: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

Show MeSH
Related in: MedlinePlus