Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

我们的工作重点是阐明：i)人类P-gp的催化循环和转运途径；ii) P-gp多特异性的分子基础；iii)临床相关酪氨酸激酶抑制剂（TKIs）与P-gp和ABCG2的相互作用；iv)测定第二代TKI尼罗替尼在P-gp上的结合位点；v)尼罗替尼与P-gp和ABCG2结合所需的药效团特征；(6)利用单粒子低温电镜分析人P-gp在催化循环过程中的构象景观。我们一直在使用基于细胞的、生物化学的、生物物理的、药理学的和生理学的技术，以及分子生物学和分子建模方法来扩展我们对ABC药物转运体的机制方面和结构-功能关系的理解。此外，我们还在P-gp和ABCG2的TKIs和小分子调节剂的筛选和开发方面投入了大量的精力。我们发现临床上用于治疗各种类型癌症的几种酪氨酸激酶抑制剂是P-gp和/或ABCG2的转运底物或抑制剂。1. 阐明ATP水解的催化循环和P-gp转运途径以及保守基序在ATP结合盒中的作用：我们正在继续对P-gp的催化循环和转运途径进行研究。我们正在使用分子模型和诱变方法在分子水平上阐明这种转运体如何识别和运输各种结构不同的化合物。我们已经开始使用tmFRET，这是一种新的生物物理方法，用于在非常低的浓度下确定蛋白质不同位置的短距离（5 - 25埃）距离。使用这种基于荧光的灵敏方法，我们已经开始确定与载脂蛋白和P-gp的封闭（ATP/Vi捕获）构象相关的距离变化。利用tmFRET，初步结果表明两个nbd在载子构象和闭合构象（20埃）之间的距离有显著变化。同样，与氧化剂菲罗啉铜和双功能巯基试剂的二硫交联研究结果表明，人P-gp是一种非常灵活的分子，其ndb以载脂蛋白形式彼此更接近。NBDs的Walker A基序中C431和C1074残基之间的距离在载子构象中为5 - 25埃（在没有ATP和药物底物的情况下）。在与法国里昂大学CNRS的Di Pietro博士的团队合作中，我们发现人类ABCG2的连接区域含有另一个C-特征基序，该基序的前两个残基（L和S）的点突变完全消除了运输偶联atp酶的活性，并导致细胞对米托蒽醌的抗性完全逆转。这些结果表明，ABCG2连接区的c2特征基序在ATP结合和/或该转运体的水解中起重要的机制作用。2. 药物介导的P-gp atp酶活性抑制机制：P-gp的大多数底物或调节剂刺激其基础atp酶活性，仅发现少数药物抑制其活性。Zosuquidar， tariquidar和elacridar是高亲和力的转运功能抑制剂，也抑制Pgp ATP酶活性，而各种底物包括维拉帕米，紫杉醇和长春花碱刺激ATP水解。在这两种情况下（刺激或抑制）起作用的分子机制仍然难以捉摸。开发有效的P-gp抑制剂肯定会受益于对药物介导的ATP水解抑制的理解。通过定向诱变，我们鉴定了P-gp的一对苯丙氨酸-酪氨酸结构基序，它们对高亲和调节剂抑制ATP水解至关重要。这些结构基序位于Pgp的药物结合口袋中。我们发现，当这些残基中的任何一个发生突变时，抑制atp酶活性的药物会转变为刺激atp酶活性。例如，zosuquidar以高亲和力抑制无cyysless WT P-gp的基础ATP水解（50%抑制所需浓度= 10-20 nM）。当Y953突变为丙氨酸时，抑制作用完全消失，当三个极性残基突变（Y307A/Q725A/Y953A）时，抑制作用转变为刺激作用。分子模拟结果表明，苯丙氨酸残基F978和F728分别与酪氨酸残基Y953和Y310呈边对面相互作用，有助于酪氨酸残基选择合适的取向，有效地与抑制剂建立氢键接触。生化研究和完整细胞的转运研究表明，抑制剂在高亲和力位点结合，产生ATP水解和转运的抑制。突变后，它们结合在较低的亲和力位点，导致ATP水解刺激和运输抑制不良。这些研究还表明，筛选能够抑制基础atp酶活性的化合物可以作为鉴定P-gp高亲和力调节剂的可靠工具。3. 人类Pgp三维结构的分辨率：P-gp三维结构的分辨率是一个正在进行的项目，为此我们开发了一种纯化方案，产生了7.5-10.0 mg的99%均质纯Pgp总蛋白。由于人体P-gp的柔韧性和产生良好衍射质量的晶体的困难，我们也使用低温电子显微镜技术进行单粒子分析。目前的研究表明，在9.5 ~ 13埃的分辨率下，可以观察到人Pgp在构象敏感单克隆抗体Fab存在和不存在时的结构特征。我们惊讶地发现，在缺乏转运底物和核苷酸的情况下，人类P-gp可以以开放（nbd分开，向内）和封闭（nbd关闭，向外）的构象存在。这些研究进一步证明了P-gp在钒酸盐和ATP （adp -钒酸捕获）存在下的nbd相互接近。而单独存在ADP时，nbd之间存在不同程度的分离。我们正在优化条件以达到亚纳米分辨率，以获得至少三种不同构象（载脂蛋白，adp -钒酸盐捕获和fab结合）的人体P-gp结构。冷冻电镜研究是与Sriram Subramanian博士合作进行的。ABCG2上的底物和调制器结合位点：关于ABCG2上的底物和调制器结合位点的信息非常有限。我们已经开始使用诱变和分子建模方法来确定底物和调节剂结合位点。由于ABCG2是一种同型二聚体，底物和调节剂的结合位点可能位于两个单体的间期。我们发现10 -组氨酸标签在n端或c端的位置对转运体的功能有影响。虽然带有10 -组氨酸标签的ABCG2在细胞表面表达良好，但其转运功能完全被取消。另一方面，n末端的10 -组氨酸标签对表达水平和转运功能没有影响，这与没有任何标签的野生型蛋白相似。c端组氨酸标签干扰了ABCG2的催化循环。

We have focused our efforts to elucidate: i) the catalytic cycle and transport pathway of human P-gp; ii) the molecular basis of the polyspecificity of P-gp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) with P-gp and ABCG2; iv) determination of the binding site of nilotinib, a second generation TKI, on P-gp; v) pharmacophore features required for binding of nilotinib to P-gp and ABCG2; and vi) the use of single particle cryo-EM for the analysis of the conformational landscape of human P-gp during its catalytic cycle. We have been employing cell-based, biochemical, biophysical, pharmacological, and physiological techniques along with molecular biology and molecular modeling approaches to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we have devoted considerable effort to the screening and development of TKIs and small molecule modulators for P-gp and ABCG2. We found that several tyrosine kinase inhibitors, which are used in the clinic for treatment of various types of cancers, are either transport substrates or inhibitors of P-gp and/or ABCG2. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of P-gp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of P-gp. We are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. We have begun to use tmFRET, which is a novel biophysical method developed to determine short range (5 - 25 angstrom) distances within different locations of the protein at very low concentrations. Using this sensitive fluorescence-based method, we have begun to determine the changes in distance associated with the apo and the closed (ATP/Vi trapped) conformations of P-gp. With tmFRET, preliminary results show that there is a significant change in the distance of the two NBDs between the apo and closed conformations ( 20 angstrom). Similarly, results of disulfide crosslinking studies with the oxidant copper phenanthroline and bi-functional sulfhydryl group reagents indicate that human P-gp is a very flexible molecule and its NBDs are much closer to each other in the apo form. The distance between the C431 and C1074 residues in the Walker A motif of NBDs ranges from 5 to 25 angstroms in the apo conformation (in the absence of ATP and drug-substrate). In collaboration with Dr. Di Pietro's group at the CNRS, University of Lyon, France, we found that the linker region of human ABCG2 harbors another C- signature motif and point mutations of the first two residues (L and S) of this motif completely abolished the transport-coupled ATPase activity, and resulted in complete reversal of cell resistance to mitoxantrone. These results suggest that the C2-signature motif in the linker region of ABCG2 plays an important mechanistic role in ATP binding and/or hydrolysis of this transporter. 2. Mechanism of the drug-mediated inhibition of P-gp ATPase activity: Most of the substrates or modulators of P-gp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Zosuquidar, tariquidar and elacridar, high affinity inhibitors of transport function, also inhibit Pgp ATPase activity, while a variety of substrates including verapamil, paclitaxel and vinblastine stimulate ATP hydrolysis. The molecular mechanisms that are in play, in either case (stimulation or inhibition), remain elusive. The development of an effective P-gp inhibitor certainly would benefit from the understanding of drug-mediated inhibition of ATP hydrolysis. Using directed mutagenesis, we identified a pair of phenylalanine-tyrosine structural motifs of P-gp that are critical for the inhibition of ATP hydrolysis by high-affinity modulators. These structural motifs are located in the drug-binding pocket of Pgp. We found that drugs that inhibit the ATPase activity switch to stimulating the ATPase activity when any of these residues are mutated. For instance, zosuquidar inhibits the basal ATP hydrolysis of cysless WT P-gp with high affinity (concentration required for 50 percent inhibition = 10-20 nM). The inhibition is completely lost upon mutation of Y953 to alanine and is switched to stimulation when three polar residues are mutated (Y307A/Q725A/Y953A). Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with the tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, helping the tyrosine residues to adopt the proper orientation to effectively establish hydrogen-bond contact with the inhibitors. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport. Upon mutation, they bind at lower affinity sites that lead to stimulation of ATP hydrolysis and a poor inhibition of transport. These studies also demonstrated that screening chemical compounds for their ability to inhibit the basal ATPase activity can be a reliable tool to identify modulators with high affinity for P-gp. 3. Resolution of the three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of P-gp is an ongoing project and for this we have developed a purification scheme that has yielded total protein of 7.5-10.0 mg of 99% homogeneously pure Pgp. Due to the flexible nature of human P-gp and the difficulty of generating crystals of good diffraction quality, we are also using single particle analysis by the cryo-electron microscopy technique. The current studies indicate that the structural features of human Pgp in the presence and absence of a Fab of conformation-sensitive monoclonal antibody can be observed at 9.5 to 13-angstrom resolution. We surprisingly found that in the absence of the transport substrate and nucleotides, human P-gp can exist in both open (NBDs apart; inward-facing) and closed (NBDs close; outward facing) conformations. These studies further demonstrated that the NBDs of P-gp in the presence of vanadate and ATP (ADP-vanadate trapped) were found to be close to each other. Whereas in the presence of ADP alone, the NBDs were separated from each other to varying degrees. We are optimizing conditions to reach a sub-nanometer resolution to obtain the structure of human P-gp in at least three different (apo, ADP-vanadate trapped and Fab-bound) conformations. The cryo-EM studies are carried out in collaboration with Dr. Sriram Subramanian. 4. Substrate and modulator-binding sites on ABCG2: There is very limited information available on the substrate and modulator binding sites on ABCG2. We have begun by using a mutagenesis and molecular modeling approach to identify substrate and modulator binding sites. As ABCG2 is a homodimer, it is possible that the substrate and modulator binding sites are located at the interphase of two monomers. We found that the position of the ten-histidine tag at the N-terminal or the C-terminal end has an effect on the function of the transporter. Although the ABCG2 with the ten-histidine tag is expressed well at the cell surface, it's transport function is completely abolished. On the other hand, the ten-histidine tag at the N-terminal has no effect on the expression level or transport function, which is similar to the wild-type protein without any tag. It appears that the histidine tag at the C-terminal interferes with the catalytic cycle of ABCG2.