Structural Studies of Alix and ESCRT Complexes in HIV-1 Budding

HIV-1出芽过程中阿利克斯和ESCRT复合物的结构研究 HIV-1颗粒的组装和释放依赖于一个蛋白质网络，包括阿利克斯和Vps 4A/B，以及四种多蛋白复合物：Hrs/STAM和ESCRT-I、II和III。这些蛋白质和复合物从酵母到人类都是保守的，它们的正常功能是将单泛素化受体、酶和其他货物分类到溶酶体或液泡。在这个网络中测试的几种蛋白质中的任何一种失活都会阻止HIV的释放和感染性。该项目的目标是：1）在单个蛋白质结构域水平上绘制HIV和ESCRT组分之间的分子相互作用; 2）表征这些相互作用中涉及的组分的结合亲和力和相关结构; 3）与NCI的Eric Freed合作，设计抑制HIV-1从细胞中出芽的方法。 HIV-1的出芽需要Gag p6蛋白的PTAP晚期结构域与ESCRT-1的TSG 101亚基的UEV结构域结合。这种基序在细胞中的正常功能是下调受体。在本财年，我们报告了1.4至1.6结构的人TSG 101 UEV结构域单独和野生型和突变型HIV-1 PTAP和Hrs PSAP九肽。P（S/T）AP基序中Thr或Ser残基的羟基与Asn 69的主链形成氢键。将Asn突变为Pro，阻断主链酰胺，在体外消除PTAP基序结合并阻断HIV-1从细胞出芽。TSG 101的N69 P和其他PTAP结合缺陷等位基因不能挽救HIV-1出芽。然而，突变等位基因确实挽救了内源性EGF受体的下调。这表明PSAP基序在正常条件下在EGF受体下调中不是速率决定性的。我们继续确定修饰的肽模拟物的结构，并使用这些结构来解释额外设计的相互作用的亲和力增益。 ESCRT-0和ESCRT-I复合物的正常功能是协调泛素化货物的聚集与内体膜的腔内出芽，这是从酵母到人类的空泡/溶酶体蛋白分选中的两个基本步骤。与上述肽结合的人TSG 101 UEV结构域的结构导致了关于UEV结构域进化的难题。酵母ESCRT-0和ESCRT-I复合物的相互作用区域的1.85晶体结构揭示了Vps 27 ESCRT-0亚基的PSDP基序结合到以Trp 16为中心的ESCRT-I亚基Vps 23的UEV结构域上的新的正电性N-末端位点。这个新的位点与人UEV结构域的C-末端部分完全不同，所述C-末端部分结合人ESCRT-0和HIV-1 Gag的P（S/T）AP基序。新PSDP结合位点的破坏消除了体外相互作用，并阻断了酵母细胞内体相关E类区室中Vps 23的富集。然而，该位点对于ESCRT货物Cps 1的分选不是必需的。总之，这些结果显示了保守的基序/结构域对如何在不同的生物体中进化成使用截然不同的结合模式。

Structural Studies of Alix and ESCRT Complexes in HIV-1 Budding HIV-1 particle assembly and release depend on a protein network that includes Alix and Vps4A/B, and four multiprotein complexes: Hrs/STAM and ESCRT-I, II, and III. These proteins and complexes are conserved from yeast to human, and their normal function is to sort monoubiquitinated receptors, enzymes, and other cargo to the lysosome or vacuole. Inactivation of any one of several proteins tested in this network blocks HIV release and infectivity. The objectives of this projects are to 1) map the molecular interactions between HIV and ESCRT components at the level of individual protein domains; 2) characterize the binding affinities and relevant structures of the components involved in these interactions; and 3) in collaboration with Eric Freed, NCI, to design means for inhibiting HIV-1 budding from cells. Budding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. In this FY we reported the 1.4 to 1.6 structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main-chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions. We went on to determine the structure of modified peptidomimetics and used the structures to explain the gain in affinity from additional designed-in interactions. The normal function of the ESCRT-0 and ESCRT-I complexes is to coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The structures of the human TSG101 UEV domain bound to the peptides described above led to a conundrum about the evolution of the UEV domain. The 1.85 crystal structure of interacting regions of the yeast ESCRT-0 and ESCRT-I complexes reveals that PSDP motifs of the Vps27 ESCRT-0 subunit bind to a novel electropositive N-terminal site on the UEV domain of the ESCRT-I subunit Vps23 centered on Trp16. This novel site is completely different from the C-terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT-0 and HIV-1 Gag. Disruption of the novel PSDP binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome-related class E compartments in yeast cells. However, this site is nonessential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms.