Dynamics of the Lactose Permease of Escherichia Coli

描述（由申请人提供）：我们的目标是通过大肠杆菌的乳糖渗透酶（LacY）了解乳糖/H+同调的动力学，LacY是主要促进剂超家族的一个范例，它包含例如囊泡单胺转运蛋白（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，世界上最广泛使用的两种药物[血清素选择性再摄取抑制剂（SSRIs）和胃质子泵抑制剂（PPIs）]是针对膜转运蛋白的。野生型LacY的近原子水平结构、构象限制突变体、单cys突变体和许多其他突变体的文库为LacY的结构和机制提供了重要信息。该蛋白由两个伪对称的6个跨膜螺旋束组成，大部分形状不规则，围绕着一个仅向细胞质一侧开放的大的亲水内腔。外质侧被紧密包裹，因此糖和氢离子的结合位点无法从这一侧进入。这种结构先验地导致了这样一种观点，即该机制涉及到一个整体的构象变化，在这个过程中，向内的空腔关闭，打开了一个质周通路，这样结合位点就可以从膜的两侧交替进入（即交替进入模型）。虽然这些结构揭示了许多新的观察结果，并证实了许多发现，但我们刚刚开始深入了解LacY在配体结合和周转过程中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腔的打开和关闭速率以及重构和H+电化学梯度的影响等基本问题。将这些发现与现有数据相结合，将有助于更深入地了解半乳糖苷/H+同调的机制，并对膜运输的重要领域产生更大的影响。

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.