STRUCTURAL BIOLOGY OF MACROMOLECULAR COMPLEXES

大分子复合物的结构生物学

• 批准号: 6100389
• 负责人: ALASDAIR C. STEVEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6100389
• 关键词: active sites; adenosinetriphosphatase; bioenergetics; biomechanics; conformation; crosslink; endopeptidases; enzyme substrate complex; fibrous protein; image enhancement; keratinocyte; membrane proteins; protein folding; protein structure function; scanning transmission electron microscopy; structural biology; transmission electron microscopy; virus protein

Background. Many important cellular functions are performed not by individual molecules but by large macromolecular complexes. The goal of this project is to elucidate the structures, assembly properties, and interactions of such complexes with emphasis on their functional connotations. We are currently working on four kinds of complexes. 1) Cornified cell envelopes (CEs) are covalently cross-linked layers of protein that line the cytoplasmic surface of terminally differentiated keratinocytes in the epidermis and other squamous stratifying epithelia. CEs are thought to play a major role in conferring the physical resilience and impenetrability of these tissues. We have a long-term interest in the biogenesis of CEs, whose covalently cross-linked character has thwarted conventional biochemical approaches. In previous work, we used immunocytochemistry to demonstrate that loricrin and the small proline-rich proteins (SPRs) are integral components of the CE and that the CE is assembled from dispersed precursors as granulocytes mature into corneocytes. We also developed a mathematical modeling approach for amino acid composition data to estimate the protein composition of CEs, and found that loricrin is the major component (60 ? 80%). Two main lines of enquiry were pursued over the past year. (a) One employs a novel method of mapping of sulfur in thin sections by electron energy loss spectroscopy and element- specific electron imaging. Loricrin has a high content of cysteine (and hence, of sulfur) and thus may be mapped by this technique. Preliminary results, outlined in last year?s report, have been completed and published. Sulfur was detected in round granules in the granulocyte cytoplasm and at the periphery of corneocytes, supporting our proposal that loricrin redistributes from these granules into the nascent CE. The amounts of sulfur detected imply that L-granules consist exclusively of loricrin, or that any additional proteins have an equally high sulfur content. These data also confirmed the large fraction of loricrin in the CE, previously estimated from its amino-acid composition. (b) Loricrin Monolayer Model of the CE. We measured the thickness and density (mass-per-unit-area) of mature CEs isolated from newborn mouse epidermis, forestomach, and footpad, respectively. Their thicknesses were measured from metal-shadowed specimens, and their mass-per-unit-area by dark-field scanning transmission electron microscopy. The CEs were found to be strikingly uniform in both thickness (14.7 nm) and mass-per-unit- area (7.2 kDa/nm2), regardless of source. Based on these data, we formulated a model in which the CE is envisaged to consist of a sheet of elongated (12nm) loricrin molecules, packed side-by- side, with their long axes perpendicular to the plane of the CE. Loricrin is cross-linked by SPRs and attached to a scaffold of involucrin and other minor CE components. This model satisfies all current data and accounts for the remarkable uniformity of the CE, which is otherwise difficult to explain. Our continuing work on the CE applies similar methods to CEs from several sources ? from cultured cells; immature CEs isolated from epidermis; and CEs from loricrin knockout mice created by our colleagues at Baylor. (2) A major portion of intracellular proteolysis in bacteria and eukaryotes alike is carried out by energy-dependent proteases, which generically consist of a proteolytic component and an ATP- hydrolyzing component. We have focused on the ClpAP enzyme of E. coli, an attractive model system. Our earlier work, based on negative staining electron microscopy and two-dimensional image analysis showed that the protease, ClpP, consists of two apposed heptameric rings of 21kDa subunits, and the ATPase, ClpA, is a single hexameric ring of 84-kDa subunits. We found that ClpA stacks axially on one or both faces of ClpP to form active complexes, whose digestion chamber lies inside ClpP. The current paradigm ? to which these observations made a major contribution ? is that ClpA recognizes substrates, unfolds them, and feeds them into the digestion chamber. Our main new results relate to a full three-dimensional account of ClpAP complexes preserved in their native states in vitreous ice. In this analysis, we characterized the symmetry mismatch that occurs when 6-fold ClpA is interfaced with 7-fold ClpP, by identifying the particular angles at which both components were viewed in micrographs of individual complexes. These observations, which made use of the recently solved crystal structure of ClpP, led to a proposal that incremental relative rotation takes place between ClpP and ClpA during processive digestion of substrates. This study has now been prepared for publication and our continuing work is aimed at a more detailed characterization of the conformational changes that we have observed to affect ClpA on binding ATP and substrates. We are also examining other related enzymes, such as Lon, in which both activities occur in the same protein subunit. 3) In a new project, we studied the binding of myosin 1C from Acanthamoeba (AMIC) - a single-headed, non-filamentous, myosin - to actin filaments by cryo-electron microscopy. The goal of this project is to characterize the structural correlates of regulation of AMIC?s ATPase activity by heavy chain phosphorylation and of ADP binding to AMIC. For myosins to function as actin-activated ATPases, a specific residue ? serine- 329, in the case of AMIC - must be phosphorylated. However, constitutively active and inactive AMIC molecules were obtained by substituting this Ser with Glu or Ala, respectively. These mutants were found to be indistinguishable in their mode of binding to actin, implying that the regulatory event affects some other aspect of the force-transducing cycle. We also investigated whether, in the presence of ADP, AMIC assumes a different conformation on the actin filament from its rigor state, as has been reported for two other myosins. We find that any such difference is very slight, a conclusion that correlates with kinetic differences that that have been detected between AMIC and the myosins that exhibit ADP-induced structural changes.

背景。许多重要的细胞功能是 不是由单个分子而是由大分子进行 复合物。该项目的目标是阐明结构， 组装特性以及此类复合物与的相互作用 强调其功能内涵。我们目前 致力于四种综合体的研究。 1) 角质化细胞膜 （CE）是共价交联的蛋白质层，排列在 终末分化角质形成细胞的细胞质表面 表皮和其他鳞状复层上皮。 CE被认为 在赋予身体弹性和 这些组织的不可渗透性。我们对以下领域有长期兴趣 CE 的生物发生，其共价交联特征 阻碍了传统的生化方法。在之前的工作中， 我们使用免疫细胞化学来证明兜甲素和 富含脯氨酸的小蛋白 (SPR) 是 CE 并且 CE 由分散的前体组装而成 粒细胞成熟为角质细胞。我们还开发了一个 氨基酸组成数据的数学建模方法 估计CE的蛋白质组成，发现兜甲素 是主要成分 (60 ? 80%)。两条主要询问线 在过去的一年里受到了追捕。 (a) 采用一种新颖的方法 通过电子能量损失在薄片中绘制硫图 光谱学和元素特异性电子成像。 Loricrin 有一个 半胱氨酸含量高（因此硫含量高），因此可能是 通过这种技术进行映射。初步结果，概述于最后 年度报告已完成并发布。硫磺是 在粒细胞胞质中和在粒细胞胞浆中检测到圆形颗粒 角质细胞的外围，支持我们的建议，即兜甲素 从这些颗粒重新分配到新生的 CE 中。金额 检测到的硫暗示 L-颗粒仅由 兜甲素，或任何其他蛋白质都具有同样高的硫 内容。这些数据也证实了兜甲素的很大一部分 CE，先前根据其氨基酸组成估计。 (二) CE 的 Loricrin 单层模型。我们测量了厚度 和从分离的成熟CE的密度（每单位面积的质量） 分别为新生小鼠表皮、前胃和足垫。 它们的厚度是根据金属阴影样本测量的， 及其通过暗场扫描传输得到的单位面积质量 电子显微镜。发现 CE 非常均匀 厚度 (14.7 nm) 和单位面积质量 (7.2 kDa/nm2)， 无论来源如何。根据这些数据，我们制定了一个模型 其中CE被设想为由一张细长的片材组成 (12nm) 兜甲素分子，并排排列，其长 轴垂直于 CE 平面。 Loricrin 是交联的 通过 SPR 并附着在外皮蛋白和其他次要分子的支架上 CE 组件。该模型满足当前所有数据和账户 CE 的显着均匀性，否则很难 解释一下。我们对 CE 的持续工作采用了类似的方法 来自多个来源的CE？来自培养细胞；不成熟的CE 与表皮分离；和来自 loricrin 敲除小鼠的 CE 由我们贝勒的同事创建。 (2) 大部分 细菌和真核生物中都进行细胞内蛋白水解作用 由能量依赖性蛋白酶产生，通常由 蛋白水解成分和 ATP 水解成分。我们 重点研究了大肠杆菌的 ClpAP 酶，这是一个有吸引力的模型 系统。我们早期的工作基于负染色电子 显微镜和二维图像分析表明 蛋白酶 ClpP 由两个并置的 21kDa 七聚环组成 亚基，而 ATP 酶 ClpA 是一个六聚环 84 kDa 亚基。我们发现 ClpA 在一个或两个上轴向堆叠 ClpP 的表面形成活性复合物，其消化室 位于 ClpP 内部。当前的范式？这些 观察做出了重大贡献？ ClpA 认识到 底物，展开它们，并将它们送入消化 室。我们的主要新成果涉及完整的三维 保存在其天然状态下的 ClpAP 复合物的说明 玻璃质冰。在此分析中，我们表征了对称性 当 6 倍 ClpA 与 7 倍连接时发生错配 ClpP，通过识别两个组件的特定角度 在单个复合物的显微照片中观察到。这些 观测，利用了最近解决的晶体 ClpP 的结构，导致了一项提议，即增量相对 在进行过程中，ClpP 和 ClpA 之间发生旋转 底物的消化。目前该研究已准备好 出版物和我们持续的工作旨在更详细地 我们所具有的构象变化的表征 观察到影响 ClpA 结合 ATP 和底物。我们是 还检查其他相关酶，例如 Lon，其中两者 活性发生在同一蛋白质亚基中。 3）在一个新项目中，我们 研究了来自棘阿米巴 (AMIC) 的肌球蛋白 1C 的结合 - a 单头、非丝状、肌球蛋白 - 至肌动蛋白丝 冷冻电子显微镜。该项目的目标是表征 AMIC ATP酶活性调节的结构相关性 通过重链磷酸化和 ADP 与 AMIC 的结合。为了 肌球蛋白作为肌动蛋白激活的 ATP 酶发挥作用，是一个特定的残基？ 丝氨酸- 329，就 AMIC 而言 - 必须被磷酸化。 然而，组成型活性和非活性 AMIC 分子 通过分别用Glu或Ala取代该Ser而获得。 发现这些突变体的模式无法区分 与肌动蛋白结合，意味着调控事件影响某些 力转换循环的其他方面。我们还调查了 在 ADP 存在的情况下，AMIC 是否会假设不同的情况 肌动蛋白丝的构象从其严格状态开始，如前所述 报道了另外两种肌球蛋白。我们发现任何这样的差异都是 非常轻微，与动力学差异相关的结论 已在 AMIC 和肌球蛋白之间检测到 表现出 ADP 诱导的结构变化。