Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

• 批准号: 6559198
• 负责人: DI S XIA
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/6559198
• 关键词: P glycoprotein; bacterial proteins; chemical aggregate; chemical models; computer simulation; cytochromes; protein protein interaction; protein structure function; structural biology

Recent advances in genome research have provided new insight into the importance of membrane protein in all cells. In most eukaryotic organisms, over one-third of the open reading frames are predicted to be integral membrane proteins with anywhere from one to fourteen transmembrane segments. Membrane proteins provide many vital cellular functions including cell-cell communication such as recognition, adhesion, and membrane fusion; in material exchange including transportation and detoxication; and in processes of cellular energy conservation. High-resolution structural studies on a limited number of membrane proteins have contributed to our understanding of function of these biological macromolecules. The demand for structural knowledge of membrane proteins has increased more than ever in light of an increased number of membrane proteins for which important functions have been identified. However structural data on membrane proteins at atomic resolution is only being obtained rather slowly, mainly due to the tremendous difficulty in purifying sufficient quantity of membrane proteins, especially those of eukaryotic origin. There is a great need for improved methods of over-producing membrane protein and of producing diffraction quality membrane protein crystals. We, in collaboration with both intramural and extramural laboratories, explore the structure and function relationships of polytopic membrane proteins crystallographically by examining a few carefully selected membrane proteins. Our efforts are concentrated on those involved in cellular multidrug resistance (human P-glycoprotein) and respiration (cytochrome bc1complex of both mitochondria and bacteria). These studies have resulted in a better understanding of membrane protein architecture in general and of the mechanism of function of these important biological system in particular. It is hoped that our continued studies will aid in the development reagents with potential therapeutic value. Another area of interest is multicomponent protein complexes, such as those involved in regulated protein degradation. Cellular regulation requires protein remodeling activities, which affect intracellular protein degradation, folding, quality control, docking and interaction. One important aspect of protein remodeling is ATP-dependent protein degradation. Almost all important cytosolic and nuclear protein degradation is carried out by ATP dependent proteases, which often consist of a hexameric chaperone and either a hexameric or a heptameric protease and have been found in all organisms. The ClpAP protease, which we have begun to study crystallographically, is especially important for a number of reasons. First, ClpAP or its close homolog, ClpXP, are essential in many microorganisms. Second, ClpAP and ClpXP are highly conserved: ClpAP is found in the chloroplast of all plants and in photosynthetic bacteria, and ClpXP is found in the mitochondria of eukaryotes, including humans. Third, ClpA is the prototype of the Hsp100 molecular chaperones, which include yeast Hsp104, a chaperone shown to be involved in prion formation. Fourth, ClpA has significant sequence and structural similarity to AAA proteins, a broad class of protein conformation-transducing ATPases involved in a plethora of vital cellular functions. Last, Clp and other ATP-dependent proteases are structurally and mechanistically complex proteins, whose structure/function relationships reflect important biochemical principles that need to be understood at the sub-molecular level. We have determined the crystal structure of ClpA, the regulatory component of the ClpAP complex. ClpA consists of five tandemly connected structural domains corresponding to three functional groups. The N-terminal domain represents a novel fold of a repeating motif with a pseudo two-fold symmetry. The two AAA modules (D1 and D2) are connected head-to-tail with a 90? rotation. In the crystal, ClpA subunits form a hexameric spiral in which the D1-D1 interface has considerably more electrostatic contacts than the D2-D2 interface, providing structural basis for functional segregation of the two AAA modules. A symmetric hexameric ring model of ClpA locates a potential ClpP-interaction loop on the distal surface of D2 and reveals a large negatively charged central cavity. These studies will lead to a detailed understanding of sub-domains and specific amino acid side-chains involved in substrate recognition, ATP hydrolysis and the accompanying conformationa changes, interactions leading to assembly of the chaperone and the protease complex, and the mechanism of protein unfolding, translocation, and degradation carried out by this and similar essential regulatory chaperones.

基因组研究的最新进展为所有细胞中膜蛋白的重要性提供了新的见解。在大多数真核生物中，预计超过三分之一的开放式阅读帧被视为整体膜蛋白，其中一个跨膜段从一个到1到14个。膜蛋白提供许多重要的细胞功能，包括识别，粘附和膜融合等细胞 - 细胞通信；在包括运输和解毒在内的物料交换中；并在细胞节能的过程中。关于有限数量的膜蛋白的高分辨率结构研究有助于我们理解这些生物大分子的功能。鉴于已经确定了重要功能的膜蛋白数量增加，对膜蛋白的结构知识的需求比以往任何时候都增加。然而，原子分辨率上膜蛋白上的结构数据仅得到缓慢的速度，这主要是由于净化足够数量的膜蛋白，尤其是真核生物起源的膜蛋白的巨大困难。非常需要改善产生膜蛋白和产生衍射质量膜蛋白晶体的方法。我们与壁内和壁外实验室合作，通过检查一些精心选择的膜蛋白来探索晶体学上多膜蛋白的结构和功能关系。我们的努力集中在涉及细胞多药耐药性（人P-糖蛋白）和呼吸（线粒体和细菌的细胞色素BC1COMPLEX）上。这些研究使人们对膜蛋白结构的一般程度和这些重要生物系统的功能机理有了更好的了解。希望我们的持续研究能够帮助具有潜在治疗价值的开发试剂。 感兴趣的另一个领域是多组分蛋白复合物，例如参与调节蛋白降解的蛋白质复合物。细胞调节需要蛋白质重塑活性，这些蛋白质重塑活性影响细胞内蛋白质降解，折叠，质量控制，对接和相互作用。蛋白质重塑的一个重要方面是依赖ATP的蛋白质降解。几乎所有重要的胞质和核蛋白降解都是由ATP依赖性蛋白酶进行的，ATP蛋白酶通常由六聚体伴侣组成，是六聚体或六聚体蛋白酶，并在所有生物体中都发现。由于多种原因，我们已经开始从晶体学上研究的CLPAP蛋白酶尤其重要。首先，在许多微生物中，CLPAP或其紧密同源物CLPXP是必不可少的。其次，CLPAP和CLPXP是高度保守的：在所有植物的叶绿体和光合细菌的叶绿体中发现了CLPAP，在包括人类在内的真核生物的线粒体中发现了ClPXP。第三，CLPA是HSP100分子伴侣的原型，其中包括酵母HSP104，这是一种与prion形成有关的伴侣。第四，CLPA与AAA蛋白具有显着的序列和结构相似性，AAA蛋白是涉及大量重要细胞功能的广泛蛋白构象传递ATPases。最后，CLP和其他依赖ATP的蛋白酶是结构和机械上复杂的蛋白质，其结构/功能关系反映了重要的生化原理，这些原理需要在亚分子水平上可以理解。我们已经确定了CLPA的晶体结构，CLPA是CLPAP复合物的调节成分。 CLPA由五个与三个官能团相对应的串联连接的结构域组成。 N末端结构域代表具有伪两个对称性的伪对称的重复基序的新颖倍数。两个AAA模块（D1和D2）与90的从头到尾连接？旋转。在晶体中，CLPA亚基形成了一种六聚体螺旋，其中D1-D1界面比D2-D2界面具有更大的静电接触，为两个AAA模块的功能隔离提供了结构性基础。 CLPA的对称六聚体环模型在D2的远端位置定位一个潜在的CLPP交流环，并揭示了一个较大的带负电荷的中央腔。这些研究将导致对参与底物识别，ATP水解以及随附的构象的变化，相互作用，导致伴侣组装的相互作用和蛋白酶复合物的相互作用以及蛋白质的机制以及蛋白质的易位，易位，易位，和类似基本的基本调节剂的降解的机制的相互作用的相互作用，从而详细了解参与底物识别，ATP水解和随附的构象变化，相互作用的详细理解。