Structural Basis for Mechanism of Secondary Transporters

描述（由申请人提供）：我们的长期目标是获得大肠杆菌乳糖通透酶（LacY）不同构象的晶体结构，以便在原子水平上了解乳糖/H+同向转运的机制。LacY是主要促进剂超家族以及一般膜蛋白的范例。我们的第一个X-射线晶体结构的构象限制突变体的LacY（C154 G）代表了一个重大突破的第一个结构的阳离子耦合同向转运体。在过去的资助期间，我们通过解决野生型LacY的X射线结构达到3.6 E的分辨率实现了另一项突破，这一成就花了十多年的时间，需要开发一种新的通用方法-保持结合磷脂。通过这种方法，我们还提高了C154 G LacY结构的分辨率到~2.9 E的分辨率，并表明糖结合是一种诱导拟合现象。然而，所有结构都显示出相同的面向内的构象：假对称的N-和C-末端6跨膜1-螺旋束，其中大部分是不规则的，围绕着一个大的内部亲水性空腔，该空腔向细胞质侧开放，并在周质侧紧密闭合。在吡喃半乳糖苷识别和H+易位中发挥主要作用的残基聚集在腔的顶端附近，并且从周质侧不可接近。提出了一种与结构和许多生物化学/生物物理方法相一致的机制，其核心是糖和H+结合位点在膜两侧的替代可及性。尽管大量的生物化学/生物物理数据表明，运输涉及打开和关闭向内和向外的空腔，结构需要在不同的构象（S），以获得在原子水平上的机制。我们已经获得了衍射晶体的可能的候选人，接近原子模型的建设适合的分辨率。该建议的主要目的是（i）获得LacY的构象结构，而不是面向内;（ii）获得衍射到足以显示结合水的分辨率的LacY结构，结合水可能在H+易位中起直接作用。我们将联合收割机和化学修饰相结合，以诱导不同于面向内构象的构象，这是有利于结晶。所提出的结构对于理解阳离子偶联膜转运蛋白的机制将是非常宝贵的，阳离子偶联膜转运蛋白是一类在许多细胞功能中起重要作用的蛋白质，对生物学和医学具有广泛的影响。 公共卫生相关性：膜蛋白在已测序的基因组中占很大比例，尽管它们参与了许多基本的细胞功能，并且是世界上最广泛的处方药的靶点，但它们的结构严重不足。乳糖通透酶（LacY）在生理学上催化乳糖和氢原子跨细菌大肠杆菌的膜的偶联易位，代表了相关膜转运蛋白的巨大家族的众所周知的模型，其中许多是临床上重要的。LacY已被用于开发用于研究这种类型的膜转运蛋白的许多技术。然而，为了理解其作用机制，必须获得LacY的结构，而不是我们已经获得的单一形式，这就是本提案的目的。

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.