Assembly of MHC class II molecules: A structural investigation of protein-protein and protein-lipid interactions within the lipid bilayer

膜蛋白的功能取决于它们所嵌入的脂质双层内外的相互作用。虽然膜内的蛋白质-蛋白质相互作用模块被描述，但关于蛋白质跨膜结构域（TMD）与膜脂质的特异性相互作用知之甚少。膜脂质种类的巨大多样性表明，它们除了作为双层的结构组分之外，还具有特定的功能。最近的数据表明，一个新的作用，个别脂质物种，它可以作为膜蛋白活性的调节剂。一个例子是我们最近发现的一个直接相互作用的I型膜蛋白与鞘脂。采用光亲和的方法，我们已经确定了一个高度特异性的相互作用的p24，蛋白质循环的分泌途径，与一个单一的鞘磷脂物种，涉及的头部基团和C18脂肪酰基链的脂质。这种蛋白质-鞘脂相互作用的细胞、体外和计算机表征使我们能够在p24的TMD内定义脂质结合基序。对哺乳动物数据集的生物信息学分析产生了许多含有这种推定的鞘脂结合基序的蛋白质候选物。这些蛋白质之一是人MHC II类蛋白质DQ α 1，其与DQ β 1链形成异二聚体。在其负载肽的状态下，该异二聚体易位至抗原呈递细胞的质膜，以使外源肽能够呈递至CD 4阳性T辅助细胞。MHC II类DQ α和β链的跨膜结构域序列的分析揭示了α和β链中存在保守的GXXXG二聚化模块。此外，DQ α链含有假定的鞘脂结合基序，并且在β链中发现了使人联想到该鞘脂结合基序的序列。在这里，我们的目标是i）结构化的TMD异源二聚化模块，ii）调查鞘脂结合的α和β链，和iii）在结构水平上了解的分子相互作用的脂质结合。为此，我们将调查这种相互作用，使用一些生物物理方法，包括多维固态和溶液NMR光谱在磷脂双层和bicellar环境中，结合在体外和在硅片上的方法来研究TMD-脂质相互作用的个人α/β链和异二聚体α/β复合物。此外，我们还将检测在二聚化和/或脂质结合中受损的蛋白变体。总之，这些调查将有助于我们了解的贡献TMD TMD和TMD脂质相互作用的MHC II类分子的功能。

The function of membrane proteins depends on interactions both inside and outside the lipid bilayers into which they are embedded. While protein-protein interaction modules within the membrane are described, much less is known about specific interactions of protein transmembrane domains (TMDs) with membrane lipids. The huge diversity of membrane lipid species suggests that they fulfil specific functions beyond their role as structural components of the bilayer. Recent data demonstrate a novel role of individual lipid species, which can function as modulators of membrane protein activity. One example is our recent discovery of a direct interaction of a type I membrane protein with a sphingolipid. Employing a photoaffinity approach, we have identified a highly specific interaction of p24, a protein cycling in the secretory pathway, with a single sphingomyelin species, involving both the head group and the C18 fatty acyl chain of the lipid. Cellular, in vitro and in silico characterisation of this protein-sphingolipid interaction allowed us to define a lipid-binding motif within the TMD of p24. Bioinformatics analysis of a mammalian dataset resulted in a number of protein candidates containing this putative sphingolipid-binding motif. One of these proteins is the human MHC class II protein DQ alpha 1 that forms a heterodimer with the DQ beta 1 chain. In its peptide-loaded state this heterodimer is translocated to the plasma membrane of antigen-presenting cells to enable presentation of foreign peptides to CD4-positive T helper cells. Analysis of the transmembrane domain sequences of MHC class II DQ alpha and beta chains revealed the presence of a conserved GXXXG dimerisation module in both the alpha and beta chains. In addition, the DQ alpha chains contain the putative sphingolipid-binding motif, and a sequence reminiscent of this sphingolipid-binding motif is found in the beta chains. Here we aim i) to structurally characterise the TMD heterodimerisation module, ii) to investigate sphingolipid-binding of alpha and beta chains, and iii) to understand at a structural level the molecular interactions underlying lipid binding. To this end we will investigate this interaction using a number of biophysical approaches, including multidimensional solid-state and solution NMR spectroscopy in phospholipid bilayer and bicellar environments, combined with in vitro and in silico approaches to study TMD-lipid interactions of the individual alpha/beta chains and the heterodimeric alpha/beta complex. In addition, we will characterise protein variants compromised in dimerisation and/or lipid binding. Together, these investigations will contribute to our understanding of contributions of TMD-TMD and TMD-lipid interactions to the function of MHC class II molecules.