The VASP tetramerization domain is a right-handed coiled coil based on a 15-residue repeat

Abstract

The vasodilator-stimulated phosphoprotein (VASP) is a key regulator of actin dynamics. We have determined the 1.3-Å resolution crystal structure of the 45-residue-long tetramerization domain (TD) from human VASP. This domain forms a right-handed ␣-helical coiled-coil structure with a similar degree of supercoiling as found in the widespread left-handed coiled coils with heptad repeats. The basis for the right-handed geometry of VASP TD is a 15-residue repeat in its amino acid sequence, which reveals a characteristic pattern of hydrophobic residues. Hydrophobic interactions and a network of salt bridges render VASP TD highly thermostable with a melting point of 120°C. In an estimated 10% of all proteins (1), ␣-helical coiled coils are present, and they constitute a principal protein-folding motif. Between two and five ␣-helices are wound around each other, usually forming a left-handed superhelix (2, 3). The sequences of the left-handed coiled coils are characterized by heptad repeats, (abcdefg) n , where positions a and d are occupied mostly by apolar residues. The left-handed supercoiling is induced by a small but significant difference between 3.5 residues per turn in a heptad repeat and 3.64 residues per turn in an undistorted ␣-helix (2), by which the residues in the a and d positions form a hydrophobic core. Typically, coiled coils are very specific oligomers with high thermodynamic stability, which enables them to function as oligomerization domains in many proteins (4).In addition to the most common coiled coils with heptad repeats, structures with longer repeats are possible. It is worth noting that in 1951 Pauling et al. (5) mentioned the feasibility of other periodicities, including 11-residue repeats (three ␣-helical turns, 11͞3) and 15-residue repeats (15͞4). Although researchers have focused almost exclusively on coiled coils with heptad repeats (6), 11-residue repeats were found recently in the tetrabrachion protein from Staphylothermus marinus (7). Its crystallographic structure has indeed revealed a parallel fourstranded coiled coil with a slight right-handed supercoiling (8). Moreover, it was suggested that 15-residue repeats would induce a yet more pronounced right-handed supercoiling (6).Here, we report the crystal structure of the tetramerization domain (TD) from human vasodilator-stimulated phosphoprotein (VASP). This domain includes two consecutive 15-residue (quindecad) repeats, which result in a parallel right-handed coiled-coil structure with a similar degree of supercoiling as observed in the left-handed coiled coils with heptad repeats. Furthermore, we show that VASP TD is stabilized by a hydrophobic interface as well as a network of salt bridges, which yields a highly thermostable structure. Last, we discuss how the TD may function as a regulator of actin assembly. Materials and MethodsCrystallization and Structure Determination. The TD (residues 336-380) of human VASP was expressed and purified as described (9). Crystals were grown at 20°C by using the hanging-drop method. We mixed 5...

Atomic structure of the vimentin central α-helical domain and its implications for intermediate filament assembly

“…To this end, we have used the NetSurfP algorithm (http://www.cbs.dtu.dk/services/NetSurfP) (23) that estimates the probability for a particular residue to be buried within the hydrophobic core. While well-established CC prediction algorithms such as COILS (24) only consider the heptad periodicity typical for left-handed CCs, one should also look for other possibilities, including the 11-residue (hendecad) and 15-residue (quindecad) periodicities that drive the formation of a parallel α-helical bundle and a right-handed CC, respectively (25,26).…”

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

“…To thoroughly challenge the kinetic model, we first generated oligomerization mutants by replacing the C-terminal VASP coiled-coil domain that mediates tetramerization (18), by other oligomerization motifs (Fig. 1A and Table S1).…”

“…The central proline-rich region binds to the actin-binding protein profilin (PFN) and Src homology 3 (SH3) domains (14)(15)(16). The C-terminal EVH2 domain comprises the business end of the molecule and encompasses a WASPhomology 2 (WH2) G-actin-binding (GAB) domain (9,15,17), an F-actin-binding (FAB) domain (15,17), and a short C-terminal coiled-coil region mediating tetramerization (18).…”

Geometric principles in the assembly of α-helical bundles

“…This may be deduced from figure 1c, when it is noted that the number of distinct layers is equal to the number of turns of the single-start helix within the repeat of 7, 11 or 15. Examination of the VASP structure shows that one kind of layer, designated 'de' by Kühnel et al [16], involves a ring of contacts between valine and the hydrophobic moiety of lysine on adjacent helices. In contrast, the other three kinds of layer, designated a, h and l, respectively, involve compact groups of four hydrophobic residues at the centre-leucine, isoleucine, valine or phenylalanine-which are generally similar to the clusters of four shown in figure 1g.…”

“…Here, we should also mention a 4-helix bundle with right-handed overall twist formed by the human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain of Kühnel et al [16]. The parallel α-helical components have a 15-residue repeat, which was successfully predicted to support a stable bundle [2].…”