The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Abstract

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single ␣-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the fulllength predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.Myosins make up a diverse superfamily of motor proteins (1). The human genome alone contains about 40 myosin genes (2). Of these, about one-third are "conventional" myosins (i.e. the well studied myosin 2), and the rest fall into about 10 different classes. The structure, properties and functions of the majority of myosin classes are poorly characterized and have largely been inferred from sequence comparisons rather than direct experiments on purified proteins (1-3).Muscle myosin 2 dimerizes through its ␣-helical coiled-coil tail. Therefore, it is commonly assumed that any myosin will also be dimeric if it contains a region predicted to be coiled coil. This assumption is dependent on the accuracy of coiled-coil prediction programs, such as COILS (4), PAIRCOIL, or MULTICOIL (5), which are also used by protein-fold prediction sites on the Web such as SMART (6).Although myosin 10 contains a region of predicted coiled coil (Fig. 1A), and is predicted to dimerize, this has not been determined experimentally. We noticed that part of the predicted coiled-coil domain of myosin 10 is highly enriched in charged residues (Fig. 1B). The proximal region consisting of 36 residues is particularly enriched in both positively and negatively charged residues, including the a and d positions of the heptad repeat (a-g) that are canonically hydrophobic residues in coiled coils (Fig. 1B). We suspected that this highly charged sequence is unlikely to form a coiled coil (7), suggesting that this part of myosin 10 may not be able to dimerize....

Helicity of short E‐R/K peptides

“…This trend has been previously reported in the context of Ala-based peptides. 9 Knight et al 12 have hypothesized that E-R/K peptides would exhibit greater stability with E-R interactions relative to E-K interactions. The presence of the guanidinium group may enable Arg to interact simultaneously with the negative Glu residues in both directions.…”

“…This motif of four Glu residues followed by a combination of four Lys and/or Arg residues has been found in a variety of proteins, including caldesmon, 11 myosin X, and myosin VI. 12,13 It has been shown to form long (up to 30 nm), single, stable, and relatively rigid helices (persistence length ¼ 15 nm) in various proteins. [14][15][16] Experimental measurement of helicity using circular dichroism of peptides has been used in conjunction with statistical mechanics models of helixcoil transition 17,18 to develop prediction programs for the helicity of peptides.…”

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

“…The SAH domain was designated to begin at Glu-818 where the VXV and XXV chimeras were spliced and end at Glu-861, 13 residues after previously identified SAH (13) to retain more charged residues RAQQEE-AARKQRE that may contribute to the motif. The constructs were inserted into the pBiEx3-BS vector, transfected into Sf9 cells, and FLAG-purified as for myosin X.…”

“…We define the C terminus of the SAH at the end of the charged region and preceding the start of the hydrophobic heptad repeat in the bovine sequence. Thus, we define the SAH domain to be somewhat shorter than previously defined: the entire tail ending before the pleckstrin homology domains (13) Table S1. E, initiation selectivity and transport distance ratios for the swivel constructs indicate motors that favor processive runs on single filaments.…”

“…These unusual, highly charged sequences have been proposed to form isolated ␣-helices in solution. Isolated peptide fragments with the characteristic SAH domain pattern show a marked helical CD signal and thermally unfold without apparent cooperativity (13,33). Some have proposed that this ␣-helix serves as a mechanical element that extends the lever arm of myosin VI and MyoM (33,34).…”