Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites.

Abstract

Abstract. We used a series of COOH-terminaUy deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the taft of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by <10 amino acids.

Disrupting Actin-Myosin-Actin Connectivity in Airway Smooth Muscle as a Treatment for Asthma?

“…In contrast, smooth muscle myosin differs structurally from skeletal or cardiac myosin, and it may be easier to inhibit smooth muscle myosin polymerization selectively. Although we are not aware of drugs developed specifically for this purpose, monoclonal antibodies directed toward specific parts of the myosin II tail prevented or even reversed myosin polymerization in vitro (40,41). Perhaps small-molecule drugs could be designed to achieve this goal.…”

Assembly of Acanthamoeba Myosin-II Minifilaments. Definition of C-terminal Residues Required to Form Coiled-coils, Dimers, and Octamers

“…Studies using 25 different monoclonal antibodies with mapped epitopes and nine C-terminal truncations identified regions of the coiled-coil tail involved in each step of Acanthamoeba myosin-II assembly. 10,11 For example, antibodies that bind tightly in the last 37 nm of the tail or deletion of more than 100 residues at the C terminus precluded assembly. In electron micrographs of Acanthamoeba myosin-II at high concentrations of salt, the tailpiece of one myosin occasionally associates with another myosin tail w15.0 nm from the C terminus.…”

Assembly of Acanthamoeba Myosin-II Minifilaments. Model of Anti-parallel Dimers Based on EM and X-ray Diffraction of 2D and 3D Crystals

“…This shows that construct 15T crystallizes as a dimer of coiled-coils, as expected from its assembly properties determined by sedimentation equilibrium. 3 Symmetry relationships in the EM structure require that the two coiled-coils are antiparallel, as predicted by the first step in the established assembly pathway. The EM density is w130 Å long, somewhat shorter than the w150 Å observed in electron micrographs of metalshadowed dimers of whole myosin.…”

“…Sinard et al 1,2 used electron microscopy to visualize assembly intermediates and mapped important interaction sites using recombinant protein expression and monoclonal antibody binding. 3 In the accompanying paper, we show that the last 32 of 90 heptads suffice for octamer formation while constructs with the last 14 heptads form anti-parallel dimers. 4 Therefore, the distal part of the amoeba myosin-II tail contains all of the information required for assembly.…”

“…[11][12][13][14] In the case of Acanthamoeba myosin-II, the non-helical tailpiece fulfils a similar role, since assembly does not occur in the absence of the tailpiece. 2,3,15 Although the structure of skeletal muscle myosin thick filaments has been studied extensively using electron microscopy and low-angle fiber diffraction, [16][17][18][19] the 3D packing and specific interactions that direct the formation of myosin filaments are unknown. To explore the assembly mechanism of myosin filaments, we examined 2D and 3D crystals derived from a construct designated 15T that consists of the last 137 residues of Acanthamoeba myosin-II.…”