Dynamics of the Lactose Permease of Escherichia Coli

描述(由申请人提供)：我们的目标是了解乳糖/H+转运蛋白在大肠杆菌乳糖渗透酶(Lacy)中的动态变化，Lacy是一个主要促进因子超家族的范例，它包含例如囊泡单胺转运体(VMAT)，以及GLUT1，它通过血脑屏障运输葡萄糖。与通道和ABC转运蛋白一样，离子梯度偶联的膜转运蛋白也与人类生理和疾病(如抑郁症、癫痫、糖尿病、多药耐药)高度相关。同样值得注意的是，世界上至少有两种最广泛的处方药[5-羟色胺选择性再摄取抑制剂(SSRIs)和胃质子泵抑制剂(PPI)]是针对膜运输蛋白的。野生型Lacy的近原子级结构，以及构象受限突变体，以及单半胱氨酸突变体和许多其他突变体的文库，提供了关于Lacy结构和机制的关键信息。该蛋白质由两个假对称的6个跨膜螺旋组成，大多形状不规则，包围着一个只对细胞质一侧开放的大的亲水性内腔。周质侧紧密堆积，因此糖和H+结合部位不能从这一侧到达。该结构先验地导致了这样的概念，即该机制涉及到全局构象变化，其中内向的空腔随着周质途径的打开而关闭，从而结合位点变得可以从膜的两侧交替访问(即交替访问模型)。虽然这些结构揭示了一些新的观察结果并证实了许多发现，但我们才刚刚开始深入了解Lacy在配体结合和转换过程中的构象状态及其转变的动力学。在未来，我们将通过应用本实验室开发的、目前在国际上使用的基于结构的技术来解决诸如空穴的打开和关闭速度以及重构和H+电化学梯度的影响等基本问题。将这些发现与现有的数据相结合，将有助于更深入地了解半乳糖苷/H+共转运蛋白的机制，并对膜转运这一重要领域产生更大的影响。 与公共卫生相关：膜蛋白在测序的基因组中占很大比例，它们与人类生理和疾病(例如抑郁症、癫痫、糖尿病、多药耐药)高度相关，它们是主要的药物靶标[例如选择性5-羟色胺再摄取抑制剂(SSRIs)]，但我们对其分子机制的了解远远落后于可溶性蛋白。乳糖渗透酶(Lacy)是一种著名的膜转运蛋白，是10,000个相关转运蛋白家族(主要促进器超家族)的模型，其中许多蛋白在临床上具有重要意义(例如VMAT，GLUT)。本实验室取得的进展代表了我们对膜运输一般原理的理解的重大突破，我们现在开始深入了解膜两侧结合位点的交替可及性、重要构象变化的速率及其在糖/H+结合蛋白过程中的转变。

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.