CRYSTAL STRUCTURE OF E COLI RNA POLYMERASE ALPHA SUBUNIT N TERMINAL DOMAIN

大肠杆菌RNA聚合酶α亚基N末端结构域的晶体结构

• 批准号: 6667783
• 负责人: SETH A DARST
• 金额: $ 14.27万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2003-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6667783
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

Scallop S1 In order to visualize the conformational changes occurring in the myosin head during ATP hydrolysis, we are determining the structures of the native scallop myosin head fragment (S1) (produced by proteolysis) complexed in the active site with various nucleotide analogs. The current view is that different positions of the lever arm will be found. Thus far, a 2.5
data set collected at CHESS allowed us to visualize the ADP-bound form of the molecule.This structure represents the highest resolution obtained to date for a myosin S1 fragment. We have also obtained a 4.2
data set and determined the structure of the nucleotide-free state of scallop myosin S1 which corresponds to the last step of the contractile cycle. For the first time, we have been able to describe an ~35 degree tilting movement of the lever arm by comparing two states of the same myosin isoform. Moreover, we have been able to establish at the atomic level the nature of the conformational changes occurring in the motor domain (Houdusse et al., in preparation). A 3.8
native data set of scallop S1 complexed with a MgADP.Pi analog (MgADP.vanadate) has also been collected. Structure determination of these crystals should lead to the characterization of a third conformation for the myosin head, that of the pre-power stroke . Vertebrate Smooth Muscle Myosin Head Fragments In parallel with the studies on invertebrate myosin S1 prepared by proteolysis, we have obtained crystals of several subfragment-nucleotide complexes of expressed smooth muscle myosin, a low velocity but high force vertebrate muscle myosin. Using data collected at CHESS, the crystal structures of an expressed vertebrate smooth muscle myosin motor domain (MD) and a motor domain-essential light chain (ELC) complex (MDE) with a transition state analog (MgADP.AlF4-) in the active site were determined to 2.9
and 3.5
resolution, respectively. The MDE structure with an ATP analog (MgADP.BeFx) was determined to 3.6
resolution. In all three structures, the converter domain in the C-terminal region is rotated /70 from that in skeletal subfragment 1 (S1), although the presence of the ELC affects the precise position of the converter. A comparison of the lever arm positions in MDE-AlF4- and in skeletal S1 shows that a potential displacement of /13 nm can be achieved during the power stroke. The MDE-BeFx and MDE-AlF4-structures are almost identical, consistent with the fact that they both bind weakly to actin. These results imply that MgATP binding, and not hydrolysis, primes the lever arm for the power stroke (Dominguez et al., submitted). Non-Muscle Myosins Some members of the myosin superfamily are regulated by direct binding of calcium on the calmodulin subunits in their lever arm. We have grown crystals of a lever arm fragment of myosin V containing two calmodulins in the absence of calcium. A native data set to 2.9
resolution as well as Pt and Au derivative data sets to about 3.5
resolution have been collected at CHESS. We expect to determine this structure by MIR methods in the near future. The results will allow us to see how calmodulin can bind to its target on myosin under these conditions. These results will provide the first structure of the lever arm of an unconventional myosin, and are the starting point for understanding regulation in this system.

扇贝S1，以可视化构象变化 在ATP水解过程中发生在肌球蛋白头部，我们正在确定 天然扇贝肌球蛋白头部片段(S1)的结构 (由蛋白质分解产生的)在活性部位与各种不同的 核苷酸类似物。目前的观点是，不同的立场 杠杆臂将被找到。到目前为止，2.5数据集收集于 CHESS允许我们可视化分子的ADP结合形式。 结构表示迄今为止为 肌球蛋白S1片段。我们还获得了4.2分的成绩数据集和 扇贝无核苷酸状态的结构测定 肌球蛋白S1，与收缩周期的最后一步相对应。 我们第一次能够描述~35度的温度 通过比较杠杆臂的两种状态来实现杠杆臂的倾斜运动 肌球蛋白亚型。此外，我们已经能够在 原子水平上发生的构象变化的性质 运动领域(Houdusse等人，正在筹备中)。A 3.8原生数据 一组扇贝S1与一种镁钒酸镁类似物(镁钒酸)络合 也被收集起来了。这些晶体的结构测定 应该导致第三种构象的表征 肌球蛋白头部，权力前中风的那个。脊椎动物肌肉 肌球蛋白头部片段与无脊椎动物研究平行 通过蛋白水解法制备肌球蛋白S1，我们得到了肌球蛋白S1的晶体 几种表达的平滑肌的亚片段-核苷酸复合体 肌球蛋白，一种低速但高力的脊椎动物肌肉肌球蛋白。vbl.使用 在国际象棋会议上收集的数据，表达了一个 脊椎动物肌肉肌球蛋白运动域(MD)和一个马达 结构域-必需轻链(ELC)复合体(MDE) 活性中心中的状态类似物(MgADP.AlF4-)被测定为2.9 和3.5分辨率分别为。带有三磷酸腺苷的MDE结构 模拟(MgADP.BeFx)被确定为3.6决议。在这三个国家中 结构，则C-端子区中的转换器域旋转 /70与骨骼亚片段1(S1)中的相同，尽管存在 ELC会影响转换器的精确位置。一种比较 MDE-AlF4-和骨骼S1中杠杆臂位置的比较显示 在功率过程中，可以实现/13 nm的潜在位移 卒中。MDE-BeFx和MDE-AlF4-结构几乎相同， 与它们都与肌动蛋白结合较弱的事实相一致。这些 结果表明，镁三磷酸腺苷结合，而不是水解，启动杠杆 手臂的力量冲刺(多明格斯等人，提交)。非肌肉型 肌球蛋白是肌球蛋白超家族中的一些成员，受直接 钙与其杠杆臂中的钙调蛋白亚基结合。我们 已经生长出含有两个肌球蛋白V的杠杆臂片段的晶体 在没有钙的情况下，钙调素。原生数据设置为2.9 分辨率以及铂和金的导数数据集约为3.5 在国际象棋比赛中已经收集到了解决方案。我们预计将确定这一点 结构在不久的将来将采用MIR方法。结果将允许 美国将了解钙调蛋白如何在这些情况下与其在肌球蛋白上的靶点结合 条件。这些结果将提供第一个结构 一种非常规肌球蛋白的杠杆臂，是 理解这个系统中的规则。