Fig. 1: M7BP is a bona fide binding partner for myosin-7a that relieves its state of autoinhibition.

A Domain organizations of myosin-7a and the identified binding protein M7BP (Schematics are in scale with the length of their primary sequences). Numbers indicate the positions of amino acid residues. B Immunofluorescence staining of eye imaginal discs of third instar larvae with anti-myosin-7a (green) and anti-M7BP (red) antibodies. Scale bar = 5 μm in the left panel and 2 μm in the right panels of magnified images. C SDS-PAGE analysis of actin co-sedimentation assays. Fractions of supernatant (S) and pellet (P) isolated from mixtures of actin, myosin-7a, M7BP, or myosin-5 after ultracentrifugation were indicated. D Class averages from negative-stain EM images of M7BP in 150 mM salt buffer, showing an asymmetric globular structure. Scale bars = 10 nm. E, F Class averages from EM images of myosin-7a alone (E) and myosin-7a in complex with M7BP (F) in 150 mM salt buffer with ATP. Myosin-7a alone exhibits a compact, autoinhibited conformation in which the distal end of the folded tail contacts the motor domain (see orange arrow in the global average image). In the presence of equimolar M7BP, the complex exhibits a more extended conformation with concomitant loss of contact between the tail and the motor domain (orange asterisk), indicating a release of autoinhibition due to M7BP binding. Scale bars = 10 nm. G–I Interferometric single-molecule mass photometry assays. Histograms (bin width = 10 kDa) of single particle mass values (presented as molecular weight) for the indicated protein or protein complex. Lines are the Gaussian fit to the data yielding the molecular weights of samples. The small peak at ~100 kDa mass in H was attributed to multiple proteolytic products around this size that were also shown by SDS-gels in C. Insets: Zoom of the regions for larger species. Bottom: A singe frame of differential interferometric scattering images in each assay. N = 9063 M7BP molecules (G), N = 25,773 myosin-7a molecules (H), and N = 18,826 myosin-7a + M7BP complexes (I). Molecule landing events were collected over three independent experiments.