Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

15A/2005G311

STRUCTURAL ANALYSIS OF PROCESSIVE MYOSIN MOTORS(MV-S1 & MVI-S1) BY X-RAY SOLUTION SCATTERINGYasunobu SUGIMOTO1, Osamu SATO2, Shinya WATANABE2, Mitsuo IKEBE2,
1*1
Yasunori TAKEZAWA and Katsuzo WAKABAYASHI
1Division of Biophysical Engineering, School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531,
2
Japan and Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USAIntroduction
The myosin motors produce a force and movement onactin filaments using the chemical energy of hydrolysis ofATP. Myosin V is one of the unconventional myosins likeas Myosin VI. Myosin V moves toward the barbed (+)
end of actin filaments like as muscle myosins, but myosin
VI moves toward the pointed (-) end. In order to clarify
the mechanism of the opposite directional motion on theactin filaments, we have investigated the structuralchanges of the subfragmet-1 (S1) of myosin V and VI
which are related with an ATP hydrolysis using X-ray
solution scattering techniques.

solution. The results are shown in Fig. 1. Structuralmodel of MV-S1IQ2 becomes to more compact in theMgATP solution, because of the lever-arm swinging. Itseems very similar to the structural change of MII-S1.
MVI-S1 model becomes to more elongated structure,
which moves a lever arm portion in opposite direction tothat of MII-S1 and MV-S1IQ2 in the MgATP solution.
These results indicate that the directionality of myosin
motors on actin filaments closely relates to the relational
structural changes of their lever arm portion.

Experimental
Recombinant myosin V-S1 with two IQ motifs (MVS1IQ2) and myosin VI-S1 (MVI-S1) were obtained from
Sf9 cells by baculovirus expression system. MV-S1IQ2and MVI-S1 constructs were purified and collected
through a column chromatography. Purified skeletal
muscle myosin subfragment-1 (MII-S1) was also used asa reference. The X-ray solution scattering experiments
were done at 20°C at the BL15A1 using the small-angle
diffractometer. All X-ray scattering data were collected
as a function of scattering vector length (S=2sinθ/λ) witha 1D-PSD. The protein concentration (c) was varied in
the range of 2 to 7mg/ml.
Results and Discussion
The radius of gyration (Rg) values obtained fromGuinier plot clearly showed that the opposite directional
movements were related to conformational changes [1].
The Rg value of MV-S1IQ2 was ~48 Å and the Rg ofMV-S1IQ2 in the presence of MgATP decreased ~ 2 Å.
These changes were very similar to that of the MII-S1. In
contrast, MVI-S1 had the Rg values of 48 Å and 51 Å inthe condition of with and without MgATP, respectively.
These results reveal that changes in Rg come from thepositional changes of their light chain-binding regions(lever arm portions). We have carried out the modeling
analysis to investigate the myosin structures and Rg
values during hydrolysis of ATP. Svergun and hiscolleagues have developed the programs that reconstruct
the three-dimensional structure from the one-dimensional
solution scattering profile data. We have applied thisprogram to calculate the MVIQ2-S1 and MVI-S1
structures in the presence and absence of MgATP in theirFigure 1TheReconstructed
modelsof
processive myosin S1s. (a) Models of MV-S1IQ2 in theabsence (left, blue model) and presence (right, pinkmodel) of MgATP. (b) Models of MVI-S1 in the absence(left, blue model) and presence (right, pink model) ofMgATP. Crystal structures of MV-S1IQ2 and MVI-S1
are superimposed on each solution model without MgATP.
References[1] Y. Sugimoto et al., PF Activity Rep. #22B, 245 (2005).

* waka@bpe.es.osaka-u.ac.jp

Users' Report

235