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

Peter J Knight,Kavitha Thirumurugan,Yuhui Xu,Fei Wang,Arnout P Kalverda,Walter F Stafford,James R Sellers,Michelle Peckham

doi:10.1074/jbc.m504887200

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 alpha-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 full-length 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.

Highlights

Myosins make up a diverse superfamily of motor proteins [1]
The proximal region consisting of 36 residues is 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)
Our electron microscopy further shows that the majority of the predicted coiled-coil domain does not dimerize to form a tail, but it forms a novel structure that acts to lengthen the head by about 15 nm

Summary

Introduction

Myosins make up a diverse superfamily of motor proteins [1]. The human genome alone contains about 40 myosin genes [2]. The proximal region consisting of 36 residues is 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. To determine the properties of the predicted coiled-coil domain of myosin 10, we studied a purified synthetic peptide containing the proximal highly charged region to determine its structure in solution. We went on to determine the structure of the full-length predicted coiled coil of myosin 10 by electron microscopy, using a recombinant expressed myosin 10 containing the motor, calmodulin binding domains, and full-length predicted coiled coil

Methods

Results

Conclusion