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渗透酶（LacY）不同构象的晶体结构，以便在原子水平上了解乳糖/H+同调的机制。LacY是主要促进剂超家族以及一般膜蛋白的范例。我们的第一个构象限制突变体LacY （C154G）的x射线晶体结构代表了一个重大突破，作为阳离子偶联对称子的第一个结构。在过去的资助期内，我们完成了另一项突破，将野生型LacY的x射线结构解决到3.6 E的分辨率，这一成就花费了十多年的时间，需要开发一种新的通用方法-维持结合磷脂。通过这种方法，我们还将C154G LacY结构的分辨率提高到~2.9 E，并表明糖结合是一种诱导配合现象。然而，所有结构都显示相同的内向构象：假对称的N和C端6跨膜1-螺旋束，其中大多数是不规则的，围绕着一个大的内部亲水腔，向细胞质一侧开放，在质周一侧紧密关闭。在半乳糖苷识别和H+易位中起主要作用的残基聚集在腔的顶端附近，从质周侧无法进入。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+结合位点在膜两侧的可达性。尽管大量的生化/生物物理数据表明，转运涉及向内和向外的空腔的打开和关闭，但为了获得原子水平上的机制，需要不同构象的结构。我们已经获得了可能的候选者的衍射晶体，其分辨率接近于适合原子模型的建立。本提案的主要目的是：(i)获得LacY除内向外的构象结构；（ii）获得一种LacY结构，其衍射分辨率足以观察束缚水，这可能在H+易位中起直接作用。我们将结合诱变和化学修饰来诱导不同于向内的构象，这有利于结晶。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无价的。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，对生物学和医学有着广泛的影响。

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.