Ca(2+)-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy.
Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.
Narita
A
A
Department of Physics, School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
Yasunaga
T
T
Ishikawa
T
T
Mayanagi
K
K
Wakabayashi
T
T
D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D020285:Cryoelectron Microscopy; D005456:Fluorescent Dyes / Q000378:metabolism; D005583:Fourier Analysis; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal; D009218:Myosins / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D055672:Static Electricity; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D014336:Troponin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D014961:X-Ray Diffraction
D000199:Actins; D005456:Fluorescent Dyes; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins; D002118:Calcium