Dynamics of the Lactose Permease of Escherichia Coli

描述（由申请人提供）：我们旨在了解大肠杆菌乳糖通透酶（LacY）对乳糖/H+共转运的动力学，LacY是主要易化因子超家族的一个范例，包含例如囊泡单胺转运蛋白（VMAT）以及GLUT 1，其转运葡萄糖穿过血脑屏障。与通道和ABC转运蛋白一样，离子梯度偶联膜转运蛋白也与人类生理学和疾病（例如抑郁症、癫痫、糖尿病、多药耐药性）高度相关。同样值得注意的是，世界上最广泛使用的药物中至少有两种[5-羟色胺选择性再摄取抑制剂（SSRI）和胃质子泵抑制剂（PPI）]是针对膜转运蛋白的。近原子水平的野生型LacY的结构，以及构象限制的突变体，和单半胱氨酸突变体和许多其他突变体的库，提供了关键的信息，关于LacY的结构和机制。该蛋白由两个假对称的6个跨膜螺旋束组成，大多数形状不规则，围绕着一个大的亲水性内腔，仅向胞质侧开放。周质侧紧密堆积，使得糖和H+结合位点从这一侧不可接近。该结构先验地导致这样的概念，即该机制涉及全局构象变化，其中面向内的腔随着周质途径的打开而关闭，使得结合位点变得可从膜的任一侧交替地接近（即，交替访问模式）。虽然结构揭示了一些新的观察，并确认了许多发现，我们才刚刚开始深入了解动态的LacY相对于构象状态和它们的过渡配体结合和营业额。在未来，我们将解决基本的问题，如开放和关闭的空腔和重建和H+电化学梯度的影响，通过应用本实验室开发的基于结构的技术，现在国际上使用的速率。整合的结果与现有的数据将有助于更深入地了解半乳糖苷/H+同向转运的机制，并有更大的影响膜运输的重要领域。 公共卫生相关性：膜蛋白在已测序的基因组中占很大比例，它们与人类生理和疾病（如抑郁症、癫痫、糖尿病、多药耐药性）高度相关，并且它们是主要的药物靶点[如选择性5-羟色胺再摄取抑制剂（SSRIs）]，但我们对其分子机制的理解远远落后于可溶性蛋白。乳糖通透酶（LacY）是一种众所周知的膜转运蛋白，是一个超过10，000种相关转运蛋白家族（主要促进因子超家族）的模型，其中许多蛋白在临床上很重要（例如VMAT、GLUT）。该实验室取得的进展代表了我们对膜转运一般原理的理解的重大突破，我们现在开始深入了解动态变化，即结合位点交替进入膜两侧，重要构象变化的速率及其在糖/H+同向转运过程中的转变。

DESCRIPTION (provided by applicant): We aim to understand the dynamics of lactose/H+ symport by the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily that contains for example the vesicular monoamine transporter (VMAT), as well as GLUT1, which transports glucose across the blood brain barrier. Like channels and ABC transporters, ion gradient-coupled membrane transport proteins are also highly relevant to human physiology and disease (e.g. depression, epilepsy, diabetes, multidrug resistance). Also of note, at least two of the most widely prescribed drugs in the world [serotonin selective reuptake inhibitors (SSRIs) and gastric proton pump inhibitors (PPIs)], are targeted to membrane transport proteins. Near-atomic level structures of wild-type LacY, as well as a conformationally restricted mutant, and a library of single-Cys mutants and many other mutants, have provided critical information regarding the structure and mechanism of LacY. The protein consists of two pseudo-symmetrical bundles of 6 transmembrane helices, mostly irregularly shaped, surrounding a large, hydrophilic internal cavity open to the cytoplasmic side only. The periplasmic side is tightly packed so that the sugar- and H+-binding sites are inaccessible from this side. The structure leads a priori to the notion that the mechanism involves a global conformational change in which the inward- facing cavity closes with opening of a periplasmic pathway so that the binding sites become alternatively accessible from either side of the membrane (i.e., the alternating access model). Although the structures reveal a number of novel observations and confirm many findings, we are just beginning to gain insight into the dynamics of LacY with respect to conformational states and their transitions during ligand binding and turnover. In the future, we will address fundamental questions such as rates of opening and closing of the cavities and the effect of reconstitution and the H+ electrochemical gradient by applying structure-based techniques developed in this laboratory and now used internationally. Integration of the findings with data currently available will facilitate far greater insight into the mechanism of galactoside/H+ symport and have an even greater influence on the important field of membrane transport. PUBLIC HEALTH RELEVANCE: Membrane proteins represent a highly significant percentage of the genomes sequenced, they are highly relevant to human physiology and disease (e.g. depression, epilepsy, diabetes, multidrug resistance), and they are major drug targets [e.g. selective serotonin reuptake inhibitors (SSRIs)], but our understanding of their molecular mechanisms lags far behind that of soluble proteins. The lactose permease (LacY), a well-known membrane transport protein, is a model for a family of >10,000 related transport proteins (the Major Facilitator Superfamily) many of which are clinically important (e.g. VMAT, the GLUTs). The advances this laboratory has achieved represent a major breakthrough in our understanding of the general principles of membrane transport, and we are now beginning to gain insight into dynamics with respect to alternating accessibility of binding sites to either side of the membrane, rates of important conformational changes and their transitions during sugar/H+ symport.