Structural Basis for Mechanism of Secondary Transporters

二级转运蛋白机制的结构基础

• 批准号: 8097347
• 负责人: Howard Ronald KABACK
• 金额: $ 50.18万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097347
• 关键词: Affinity Labels; Bacteria; Binding; Binding Sites; Biochemical; Biology; C-terminal; Cations; Cell physiology; Chemicals; Coupled; Coupling; Crystallization; Data; Dehydration; Detergents; Development; Drug Prescriptions; Engineering; Escherichia coli; Fab Immunoglobulins; Family; Galactose; Galactosides; Goals; Grant; Hand; Heart; Hydrogen; Hydrogen Bonding; Ion Cotransport; Lactose; Ligand Binding; Medicine; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Modeling; Modification; Molecular Conformation; Molecular Models; Monoclonal Antibodies; Mutagenesis; Phospholipids; Play; Property; Proteins; Research Design; Resolution; Robotics; Roentgen Rays; Role; Screening procedure; Side; Sodium Chloride; Specificity; Structure; Sulfhydryl Compounds; Techniques; Water; affinity labeling; base; crosslink; design; genome sequencing; hydronium ion; improved; lactose permease; molecular modeling; mutant; novel strategies; periplasm; permease; public health relevance; sugar; symporter

DESCRIPTION (provided by applicant): Our long-range goal is to obtain crystal structures of different conformations of the lactose permease of Escherichia coli (LacY) in order to understand the mechanism of lactose/H+ symport at the atomic level. LacY is a paradigm for the Major Facilitator Superfamily, as well as membrane proteins in general. Our first X-ray crystal structure of a conformationally restricted mutant of LacY (C154G) represents a major breakthrough as the first structure of a cation-coupled symporter. In the past grant period, we accomplished another breakthrough by solving an x-ray structure of wild-type LacY to a resolution of 3.6 E, an accomplishment that took well over a decade and required development of a new, general approach-maintaining bound phospholipids. By this means, we also improved resolution of the C154G LacY structure to a resolution of ~2.9 E and showed that sugar binding is an induced-fit phenomenon. However, all structures display the same inward-facing conformation: pseudo-symmetrical N- and C- terminal 6 transmembrane 1-helix bundles, most of which are irregular, surrounding a large internal hydrophilic cavity open to the cytoplasmic side and tightly closed on the periplasmic side. The residues that play major roles in galactopyranoside recognition and H+ translocation are clustered near the apex of the cavity and inaccessible from the periplasmic side. A mechanism consistent with the structure and many biochemical/biophysical approaches is proposed, the heart of which is alternative accessibility of the sugar- and H+-binding sites to either side of the membrane. Despite a wealth of biochemical/biophysical data showing that transport involves opening and closing of inward- and outward-facing cavities, structures are needed in a different conformation(s) in order to obtain the mechanism at the atomic level. We have obtained diffracting crystals of likely candidates that are approaching a resolution suitable for atomic model building. The main aims of this proposal are (i) to obtain structures of conformations of LacY other than inward facing; (ii) to obtain a structure of LacY that diffracts to a resolution sufficient to visualize bound water, which may play a direct role in H+ translocation. We will combine mutagenesis and chemical modification to induce conformations different from the inward-facing conformation, which is favored by crystallization. The proposed structures will be invaluable for understanding the mechanism of cation-coupled membrane transporters, a class of proteins that plays essential roles in many cellular functions and has broad impact on biology and medicine. PUBLIC HEALTH RELEVANCE: Membrane proteins represent a very significant percentage of the genomes sequenced, and although they are involved in a multitude of essential cellular functions and are targets for the world's most widely prescribed drugs, their structures are grossly underrepresented. The lactose permease (LacY), which physiologically catalyzes the coupled translocation of lactose and a hydrogen atom across the membrane of the bacterium Escherichia coli, represents a well-known model for a huge family of related membrane transport proteins, many of which are clinically important. LacY has been used to develop numerous techniques for studying of this type of membrane transport proteins. In order to understand its mechanism of action, however, it is essential to obtain structures of LacY in more than the single form that we have obtained, which is the purpose of this proposal.

描述（由申请人提供）：我们的远程目标是获得大肠杆菌（Lacy）乳糖渗透酶不同构型的晶体结构，以了解原子水平上乳糖/H+符号的机制。蕾丝（Lacy）是主要促进剂超家族以及膜蛋白的范式。我们的第一个X射线晶体结构是固定限制的蕾丝突变体（C154G），代表了作为阳离子耦合的共物的第一个结构的主要突破。在过去的赠款期间，我们通过将野生型蕾丝的X射线结构解决为3.6 e的X射线结构，取得了另一个突破，这一成就花费了十多年，并需要开发一种新的，一般的方法维护新的磷脂。通过这种方式，我们还将C154G蕾丝结构的分辨率提高到了〜2.9 E的分辨率，并表明糖结合是一种诱导的现象。然而，所有结构都显示出相同的向内构象：伪对称的N-和C-末端6跨膜1-螺旋束，其中大多数是不规则的，围绕大型内部亲水型腔向细胞质侧开放，并紧密地闭合。在半乳吡喃糖苷识别和H+易位中起主要作用的残基聚集在腔体的顶点附近，并且无法从周质侧访问。提出了一种与结构和许多生化/生物物理方法一致的机制，其核心是糖和H+结合位点的替代性可访问性。尽管大量的生化/生物物理数据表明，运输涉及向内和向外腔的开放和关闭，但仍需要在不同的构象中进行结构，以便在原子水平上获得机制。我们已经获得了接近适合原子模型建设的分辨率的可能候选者的衍射晶体。该提案的主要目的是（i）获得除内向外的蕾丝构象结构； （ii）获得蕾丝结构，该结构衍射为足以可视化结合水的分辨率，这可能在H+易位中起着直接的作用。我们将结合诱变和化学修饰，诱导与内向构象不同的构象，这种构象受到结晶的青睐。所提出的结构对于理解阳离子耦合膜转运蛋白的机制是无价的，阳离子偶联的膜转运蛋白是在许多细胞功能中起着重要作用并对生物学和医学产生广泛影响的一类蛋白质。 公共卫生相关性：膜蛋白代表了测序的基因组中很大一部分，尽管它们参与了多种必需的细胞功能，并且是世界上规定的最广泛处方药的目标，但其结构的体现不足。乳糖渗透酶（蕾丝）在生理上催化了乳糖的耦合易位和氢原子跨过细菌大肠杆菌的膜，它代表了一个众所周知的相关膜转运蛋白家族的众所周知模型，其中许多蛋白在临床上很重要。蕾丝（Lacy）已用于开发许多用于研究这种类型的膜转运蛋白的技术。但是，为了了解其作用机理，必须以比我们获得的单一形式获得蕾丝结构，这是本提案的目的。