Structural Basis for Mechanism of Secondary Transporters

描述(申请人提供)：我们的长期目标是获得不同构象的大肠杆菌(Lacy)乳糖渗透酶的晶体结构，以便在原子水平上了解乳糖/H+结合蛋白的机制。Lacy是主要促进剂超家族的典范，也是膜蛋白的典范。我们的第一个X-射线晶体结构是一个构象受限的Lacy突变体(C154G)，它是阳离子偶联转运体的第一个结构的重大突破。在过去的资助期间，我们完成了另一项突破，解决了野生型花边的X射线结构，分辨率达到3.6E，这一成就花了十多年的时间，需要开发一种新的、通用的方法-维持结合的磷脂。通过这种方法，我们也将C154G Lacy结构的分辨率提高到~2.9E，并表明糖结合是一种诱导拟合现象。然而，所有的结构都表现出相同的向内构象：假对称的N-末端和C-末端6跨膜1-螺旋束，其中大多数是不规则的，包围着一个大的内部亲水性空腔，在细胞质一侧开放，在周质一侧紧密闭合。在半乳糖苷识别和H+转运中起主要作用的残基聚集在空腔顶端附近，不能从周质侧进入。提出了一种与结构和许多生化/生物物理方法相一致的机制，其核心是膜两侧糖和H+结合位点的替代可访问性。尽管大量的生化/生物物理数据表明，运输涉及向内和向外空腔的打开和关闭，但需要不同构象的结构(S)才能在原子水平上获得机制。我们已经获得了接近适合于原子模型建立的分辨率的可能候选者的衍射晶体。这一建议的主要目的是(I)获得Lacy的构象结构，而不是向内；(Ii)获得Lacy的结构，该结构衍射到足以显示结合水的分辨率，这可能在H+转运中起直接作用。我们将结合诱变和化学修饰来诱导不同于内向构象的构象，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非常有价值的，阳离子偶联膜转运蛋白是一类在许多细胞功能中发挥重要作用并对生物学和医学产生广泛影响的蛋白质。 与公共卫生相关：膜蛋白在测序的基因组中占很大比例，尽管它们参与了多种基本的细胞功能，也是世界上最广泛的处方药的靶标，但它们的结构严重不足。乳糖渗透酶(Lacy)在生理上催化乳糖和一个氢原子在大肠杆菌的膜上偶联转运，代表了一个相关的膜运输蛋白大家族的众所周知的模型，其中许多蛋白在临床上很重要。Lacy已经被用来开发许多研究这种类型的膜运输蛋白的技术。然而，为了了解其作用机制，有必要获得多于我们所获得的单一形式的花边结构，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.