Krueger J K, Zhi G, Stull J T, Trewhella J
Chemical Science and Technology Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Biochemistry. 1998 Oct 6;37(40):13997-4004. doi: 10.1021/bi981311d.
Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+.calmodulin (CaM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CaM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017-6023]. We report here similar scattering experiments on the CaM.MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine-triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by approximately 12 A), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM.MLCK.pRLC.AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.
此前，我们利用小角X射线散射和对比变化的中子散射技术，获得了与功能性酶——骨骼肌肌球蛋白轻链激酶（MLCK）的一种具有酶活性的截短突变体——复合的4Ca2⁺·钙调蛋白（CaM）的首个低分辨率结构。这些实验表明，与MLCK结合后，CaM会发生构象塌缩，这与CaM结合到对应于MLCK的CaM结合序列的分离肽段时所观察到的情况相同。由此表明，CaM通过诱导其CaM结合序列和自身抑制序列显著远离催化核心表面，从而解除了对激酶的抑制作用[克鲁格，J.K.，奥拉，G.A.，罗科普，S.E.，志，G.，斯图尔，J.T.，以及特里韦拉，J.（1997年）《生物化学》36卷，6017 - 6023页]。我们在此报告了对添加底物后的CaM - MLCK复合物进行的类似散射实验；三磷酸腺苷（ATP）的一种不可水解类似物，AMPPNP，以及MLCK的一种肽底物，肌球蛋白调节轻链（pRLC）的磷酸化序列。这些底物被证明会诱导复合物整体压缩。CaM和MLCK组分的质心间距缩短（约12 Å），因此与无底物的复合物相比，CaM更靠近催化位点。此外，底物结合后，CaM相对于激酶似乎发生了重新定向，导致CaM的N端序列与激酶之间产生了在无底物的复合物中未观察到的相互作用。最后，与无底物的复合物相比，在CaM - MLCK - pRLC - AMPPNP复合物中激酶自身变得更加紧凑。观察到的MLCK在底物结合时的这种压缩与环磷酸腺苷依赖性蛋白激酶结合假底物时催化裂隙关闭所产生的压缩相似。
