Further insights into calmodulin-myosin light chain kinase interaction from solution scattering and shape restoration.
Further insights into calmodulin-myosin light chain kinase interaction from solution scattering and shape restoration.
复制标题
从溶液散射和形状恢复进一步了解钙调蛋白-肌球蛋白轻链激酶相互作用。
DOI：
10.1021/bi0348664
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Trewhella,Jill
中科院分区：
文献类型：
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作者：
Heller,WilliamT;Krueger,JoannaK;Trewhella,Jill
我们已经获得了新的见解之间的相互作用的第二信使蛋白钙调素（CaM）和肌球蛋白轻链激酶从骨骼肌（skMLCK）使用小角溶液散射和形状恢复。具体来说，我们探索了2Ca 2 +-CaM−skMLCK复合物的性质，并在相同条件下将其与4Ca 2 +-CaM−skMLCK复合物进行了比较。2Ca 2+复合物已被认为是生理相关的。为了帮助数据的解释，我们开发了一种形状恢复方法，在GA_BLOCK CT中实现，该方法将其他可用方法的许多最佳功能结合到一个单一的自动化软件包中。重要的是，GA_BSCT明确地解决了逆散射问题的多个解的存在性问题，并从一组适合输入强度的形状中产生共识包络。从已知结构测量或计算的小角散射强度分布被用于测试GA_BCT，然后用于生成三种复合物的低分辨率模型：2Ca 2 +-CaM−skMLCK，4Ca 2 +-CaM−skMLCK和结合底物的4Ca 2 +-CaM−skMLCK。这些模型与蛋白质组分的高分辨率结构结合使用，以更好地理解它们之间的相互作用。在2Ca 2 +-CaM−skMLCK复合物的情况下，共有包膜与完全塌陷状态的CaM一致，其两个球状叶彼此紧密接触，而激酶的催化裂缝是开放的。4Ca 2 +-CaM-skMLCK复合物的共识包络表明，塌陷的CaM比2Ca 2+复合物中的CaM更远离skMLCK的开放催化裂缝，并且进一步表明底物与该复合物的结合导致了激酶的关闭催化裂缝，与之前的中子散射结果一致。这些结果表明，激活MLCK的钙调素只能发生在一旦钙调素是完全易位远离催化裂缝，这大概是连接到充分释放的假底物/抑制序列。我们的散射数据表明，这一步是完成时，所有四个钙结合位点加载。
We have gained new insight into the interactions between the second-messenger protein calmodulin (CaM) and myosin light chain kinase from skeletal muscle (skMLCK) using small-angle solution scattering and shape restoration. Specifically, we explored the nature of a 2Ca2+-CaM−skMLCK complex and compared it to a 4Ca2+-CaM−skMLCK complex under the same conditions. The 2Ca2+complex has been proposed to be physiologically relevant. To aid in the interpretation of the data, we developed a shape restoration approach, implemented in GA_STRUCT, that combines many of the best features of other available methods into a single, automated package. Importantly, GA_STRUCT explicitly addresses the problem of the existence of multiple solutions to the inverse scattering problem and produces a consensus envelope from a set of shapes that fit the input intensity. Small-angle scattering intensity profiles measured or calculated from known structures were used to test GA_STRUCT, which was then used to generate low-resolution models for three complexes: 2Ca2+-CaM−skMLCK, 4Ca2+-CaM−skMLCK, and 4Ca2+-CaM−skMLCK with a bound substrate. These models were used in conjunction with high-resolution structures of the protein components to better understand the interactions among them. In the case of the 2Ca2+-CaM−skMLCK complex, the consensus envelope is consistent with CaM in a fully collapsed state with its two globular lobes in close contact with each other while the catalytic cleft of the kinase is open. The consensus envelope for the 4Ca2+-CaM−skMLCK complex indicates that the collapsed CaM has swung further away from the open catalytic cleft of the skMLCK than in the 2Ca2+complex, and further that substrate binding to this complex results in closure of the kinase catalytic cleft, in agreement with previous neutron scattering results. These results indicate that activation of MLCK by CaM can only occur once CaM is fully translocated away from the catalytic cleft, which is presumably linked to full release of the pseudo-substrate/inhibitory sequence. Our scattering data indicate that this step is completed only when all four calcium binding sites are loaded.