Shimizu Toshiyuki, Ihara Kentaro, Maesaki Ryoko, Amano Mutsuki, Kaibuchi Kozo, Hakoshima Toshio
Structural Biology Laboratory, Nara Institute of Science and Technology, and CREST, Japan Science and Technology Corporation, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
J Biol Chem. 2003 Nov 14;278(46):46046-51. doi: 10.1074/jbc.M306458200. Epub 2003 Sep 3.
Rho-kinase is a serine/threonine protein kinase that regulates cytoskeletal events in cells. The enzyme activity of Rho-kinase is auto-inhibited in the free state but is activated through direct binding to the small GTPase Rho in the GTP-bound form. The crystal structure of the Rho-binding domain (RhoBD) of Rho-kinase has been determined at 1.8-A resolution by the multi-wavelength anomalous dispersion technique. The structure shows that RhoBD dimerizes to form a parallel coiled-coil with long consecutive alpha-helices extended to approximately 97 A and suggests that free Rho-kinase can also form a dimer through parallel self-association. At the middle region of the coiled-coil, the polypeptide chains are flexible and display loose "knobs-into-holes" packing of the side chains from both chains. RhoBD residues that have been shown to be critical for Rho-binding are spread in the positively charged C-terminal region. The parallel coiled-coil structure of our Rho-kinase RhoBD in the free form is different from the anti-parallel coiled-coil structure of RhoBD of protein kinase N when complexed with RhoA. Implications derived from these structural studies in relation to the mechanism of Rho-kinase activation will be addressed with previously reported experimental data.
Rho激酶是一种丝氨酸/苏氨酸蛋白激酶，可调节细胞中的细胞骨架事件。Rho激酶的酶活性在游离状态下会自动抑制，但通过与GTP结合形式的小GTP酶Rho直接结合而被激活。已通过多波长反常色散技术以1.8埃分辨率确定了Rho激酶的Rho结合域（RhoBD）的晶体结构。该结构表明，RhoBD二聚化形成平行卷曲螺旋，长连续α螺旋延伸至约97埃，这表明游离的Rho激酶也可以通过平行自缔合形成二聚体。在卷曲螺旋的中间区域，多肽链具有柔韧性，并显示出两条链的侧链松散的“旋钮-入-孔”堆积。已证明对Rho结合至关重要的RhoBD残基分布在带正电荷的C末端区域。我们的游离形式的Rho激酶RhoBD的平行卷曲螺旋结构与与RhoA复合时蛋白激酶N的RhoBD的反平行卷曲螺旋结构不同。将结合先前报道的实验数据探讨这些结构研究对Rho激酶激活机制的影响。
