Abstract
BackgroundTropomyosin 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 FindingsTropomyosin'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 α-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).ConclusionsThis, 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.
Highlights
The a-helical coiled coil is a ubiquitous protein folding and assembly motif present in proteins that participate in a wide variety of cellular functions, including muscle contraction, transcription, membrane trafficking, and metabolism [1]
We suggest that the composition, location, and distance between actin binding periods in the tropomyosin molecule contribute to the components of binding in different ways
Even though deletion of P2 or P3 results in a modest reduction in actin affinity, replacement of the Ala cluster with a canonical coiled coil interface dramatically reduces actin affinity [17], suggesting destabilization is essential for actin binding whether by direct participation of the P2-P3 region or by determining the overall molecular shape for binding the actin filament
Summary
The a-helical coiled coil is a ubiquitous protein folding and assembly motif present in proteins that participate in a wide variety of cellular functions, including muscle contraction, transcription, membrane trafficking, and metabolism [1]. The thermal unfolding of wildtype tropomyosin analyzed using DSC showed the expected two major endotherms (Figure 3A, dotted line, Table 2). In this biphasic unfolding the first endotherm corresponds to the unstable middle region of the molecule including P5 (residues 130–190) and the Cterminal half whereas the second endotherm, 56.0uC in wildtype, is the unfolding of the N-terminal ,half. In P2Shift both transitions occurred at lower temperatures (Figure 3B, dotted line). The compensatory A120L mutation in P3Shift may stabilize this region and result in the single, lower temperature transition (49.0uC) with DSC, indicative of increased cooperativity of unfolding. It is not well understood how tropomyosin binds actin
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