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: 6491106
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2002-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6491106
• 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为了可视化构象变化 发生在肌球蛋白头部在ATP水解，我们正在确定 天然扇贝肌球蛋白头部片段（S1）的结构 （由蛋白水解产生）在活性位点与各种 核苷酸类似物。 目前的观点是， 杠杆臂将被发现。到目前为止，
数据集收集于 CHESS使我们能够可视化ADP结合形式的分子。 结构表示迄今为止获得的最高分辨率， 肌球蛋白S1片段。 我们还获得了4.2
数据集和 确定了扇贝无核苷酸状态的结构 肌球蛋白S1，其对应于收缩周期的最后一步。 这是我们第一次能够描述一个~35度的 通过比较杠杆臂的两种状态来进行杠杆臂的倾斜运动 肌球蛋白同种型 此外，我们已经能够在 原子水平上的构象变化的性质发生在 运动域（Houdusse等，准备中）。 了3.8
本机数据 一组扇贝S1与MgADP.Pi类似物（MgADP.钒酸盐）复合 也被收集起来。 这些晶体的结构测定 应该导致第三构象的特征， 肌球蛋白头，即前动力中风。 脊椎动物平滑肌 肌球蛋白头部片段与无脊椎动物的研究 通过蛋白水解制备肌球蛋白S1，我们获得了 表达的平滑肌的几个亚片段-核苷酸复合物 肌球蛋白，一种低速但强力的脊椎动物肌肉肌球蛋白。 使用 在国际象棋收集的数据，晶体结构的表达 脊椎动物平滑肌肌球蛋白运动结构域（MD）和一个运动 结构域-必需轻链（ELC）复合物（MDE），带有过渡 活性中心的MgADP·AlF_4 ~-态类似物含量为2.9
和3.5
决议，分别。 带有ATP的MDE结构 类似物（MgADP·BeFx）测定为3.6
分辨率 在所有三 结构中，C端区域中的转换器域被旋转 /70，虽然存在的骨骼亚片段1（S1）， ELC影响转换器的精确位置。 的比较 在MDE-AlF 4-和骨架S1中的杠杆臂位置显示， 在功率期间可以实现± 13 nm的电势位移 中风 MDE-BeFx和MDE-AlF 4-结构几乎相同， 这与它们都与肌动蛋白弱结合的事实相一致。 这些 结果表明，MgATP结合，而不是水解，启动杠杆 用于动力冲程的臂（Dominguez等人，提交）。 非肌肉 肌球蛋白超家族的一些成员由直接的 钙离子与杠杆臂中钙调素亚基的结合。我们 已经生长了肌球蛋白V的杠杆臂片段的晶体， 在没有钙的情况下的钙调素。 原生数据设置为2.9
分辨率以及Pt和Au衍生物数据集约为3.5
决议已收集在国际象棋。 我们希望确定这一点 在不久的将来用MIR方法进行结构。 结果将允许 我们来看看钙调蛋白如何在这些条件下与肌球蛋白上的靶点结合。 条件 这些结果将提供第一个结构的 一个非传统的肌球蛋白的杠杆臂，是起点， 了解这个系统的规则。