Deciphering the design of the tropomyosin molecule

Abstract

The crystal structure at 2.0-A resolution of an 81-residue N-terminal fragment of muscle alpha-tropomyosin reveals a parallel two-stranded alpha-helical coiled-coil structure with a remarkable core. The high alanine content of the molecule is clustered into short regions where the local 2-fold symmetry is broken by a small (approximately 1.2-A) axial staggering of the helices. The joining of these regions with neighboring segments, where the helices are in axial register, gives rise to specific bends in the molecular axis. We observe such bends to be widely distributed in two-stranded alpha-helical coiled-coil proteins. This asymmetric design in a dimer of identical (or highly similar) sequences allows the tropomyosin molecule to adopt multiple bent conformations. The seven alanine clusters in the core of the complete molecule (which spans seven monomers of the actin helix) promote the semiflexible winding of the tropomyosin filament necessary for its regulatory role in muscle contraction.

Figure 1

Differences between native tropomyosin (B, C) and the Tm81 construct (E, D, A). (A, B) Sketches showing how the first 81 residues of native α-tropomyosin differ from the Tm81 fragment at their N and C termini (also see Methods). (C, D) α-Carbon drawing of the N-terminal 14 residues of the fully helical N-acetylated TmZip NMR structure (23) and of the unacetylated Tm81 crystal structure, in which residues 1 and 2 are nonhelical. (E) The nine N-terminal residues of Tm81. The coordinates are those of the final refined model. Residues C1–C9 are shown. The electron density map is shown within 2.0 Å of each atom at a contour level 1.75 σ. It was produced by using 2F_o − F_c coefficients and phases calculated from a preliminary (and fully helical) model (similar to C) early on in the refinement procedure. This “omit”-style map for residues 1 and 2 reveals their nonhelical path, which is the same for all four chains of the asymmetric unit. At the unacetylated terminus of each Tm81 chain, the Met-1 side chain contacts surface residues 5, 6, and 9, and Asp-2 stabilizes the three exposed main-chain amino groups of residues 3, 4, and 5 at the N terminus of the α-helix. Drawings are made by using povray (www.povray.org) and a version of molscript (46) modified to read maps by E. Peisach.

Figure 2

The “alanine stagger.” (A) A parallel two-stranded coiled-coil segment rich in core alanines, such as Tm81 (fragment “AB”) residues 22–36 (Left) is axially out-of-register by ≈1.2 Å and narrower by ≈2 Å compared with a canonical 2-fold symmetric segment dominated by core leucines, such as Tm81 residues 36–50 (Right). The superposition (Middle) shows that this difference in the axial and radial dispositions of the α-helical backbones in the two segments causes the relative locations of their core side chains' terminal methyl groups (i.e., the Cβ atoms of alanine residues 25 and 32 and the Cδ1 atoms of leucine residues 39 and 46) to be nearly the same (within 0.59 and 1.09 Å, respectively). (B) The terminal methyl groups in both types of core, such as Tm81 alanine 25 (Left) and leucine 39 (Right), make contacts with at most three residues in a triangular “hole” of the neighboring helix. [As published as supplemental data on the PNAS web site, www.pnas.org, a similar axial staggering of the helices occurs in this region and in the (modified) second alanine cluster of the other (“CD”) fragment of the asymmetric unit; here differences are also described in the extent of axial staggering near the modified N and C termini between the two fragments located in different crystal environments.]

Figure 3

The “alanine bend.” (A) The joining of an axially staggered coiled-coil segment, such as Tm81 (fragment “AB”) residues 28–36 (blue), and an unstaggered segment, such as residues 36–45 (red) produces a bend of the coiled-coil axis (here at ≈ residue 36) away from the locally longer α-helix (Left). The structure of this region is nearly identical in the two crystallographically independent fragments. (Similar bends also occur at ≈ residues 22 and 68 in the “CD” fragment, as shown in the supplemental data, which also describes how supercoiling results in the coiled-coil axis at the two boundaries of a staggered segment to bend in different planes.). (B) Simplified schematic of the joint.

Figure 4

Similarities and differences in the core amino acid sequences of both muscle and nonmuscle isoforms. (A) The 284-residue long sequence of chicken striated α-tropomyosin (SWISS-PROT P02559) shows that there are seven clusters of d-position alanines (highlighted by blue triangles) in addition to the relatively regular negatively charged surface residues in the α-zones (4, 5) (bold in black square, and dashes to the side). These α-zones are implicated in the binding of tropomyosin to seven actin subunits. Note that a-position β-branched residues (highlighted in red diamonds) are common in canonical coiled-coil segments (see Discussion) but rare in the alanine clusters of this and other tropomyosin isoforms. (B) Simplified schematic diagram (supercoiling not depicted) of one of many (up to 2⁷ = 128) possible discrete conformations for tropomyosin that can be produced by the alternating seven alanine (blue staggered rectangles) and canonical (red in-register rectangles) d-position clusters. The fourth and fifth “canonical” segments are unusual in not having any d-position leucines. (C) As A, but only the core sequence of native α-tropomyosin from smooth muscle of chicken gizzard (SWISS-PROT P04268) is shown. These two alternatively spliced tropomyosin isoforms are encoded by the same gene and are identical except for exon 2 (residues 39-80) and exon 9 (residues 258-284). Similar alanine clusters are encoded by exon 2 of these two tropomyosin isoforms; in contrast, the last d position alanine in exon 9 is not conserved between these isoforms, corresponding to the general divergence of the sequences in the C-terminal region (47), which is the apparent binding site (only in skeletal muscle tropomyosin) for troponin T. In general, the alanine clusters are conserved in tropomyosin isoforms encoded by different genes. (D) Many of the alanine clusters are also present in the core sequence of the shorter 248-aa residue nonmuscle human fibroblast tropomyosin (SWISS-PROT P07226), which binds only six actin subunits and is a δ tropomyosin encoded by the TPM4 gene. Note that in this figure we have not attempted to align the first six heptads of this sequence with the other sequences shown. (E) A model of the native tropomyosin molecule and filament generated from the Tm81 coordinates (see supplemental data, www.pnas.org) and a simplified model of the actin helix [drawn by Graham Johnson (fivth.com)]. The black spheres correspond to the periodic surface acidic residues of the α-zones. [The scale of a tropomyosin molecule is different from those in A–D. Note that the precise structure is unknown for the head-to-tail overlap between two consecutive tropomyosin molecules in a filament (located about two-thirds down the figure)].