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

膜蛋白的功能依赖于它们所嵌入的脂双层内外的相互作用。虽然描述了膜内的蛋白质-蛋白质相互作用模块，但对蛋白质跨膜结构域(TMD)与膜脂的特定相互作用知之甚少。膜脂物种的巨大多样性表明，它们除了作为双层的结构成分外，还发挥着特定的功能。最近的数据表明，单个脂类物种具有新的作用，可以作为膜蛋白活性的调节器。一个例子是我们最近发现的I型膜蛋白与鞘磷脂的直接相互作用。利用光亲和的方法，我们已经确定了p24的高度特异性相互作用，p24是一种在分泌途径中循环的蛋白质，它与一种单一的鞘磷脂物种发生了高度特异性的相互作用，涉及到脂质的头基和C18脂肪酰链。这种蛋白质-鞘磷脂相互作用的细胞、体外和电子计算机特征使我们能够在p24的TMD内定义一个脂结合基序。对哺乳动物数据集的生物信息学分析导致了一些候选蛋白包含这个假定的鞘磷脂结合基序。其中一种蛋白质是人类MHC II类蛋白DQα1，它与DQβ1链形成异二聚体。在它的多肽装载状态下，这种异源二聚体被转移到抗原提呈细胞的质膜上，以使异源多肽能够提呈给CD4阳性的T辅助细胞。对MHCⅡ类DQα和β链跨膜结构域序列的分析表明，在α链和β链中都存在一个保守的GXXXG二聚化模块。此外，DQ阿尔法链包含推测的鞘脂结合基序，而在β链中发现了一个序列，使人想起这个鞘脂结合基序。在这里，我们的目标是：i)从结构上表征TMD异二聚化模块；ii)研究鞘磷脂与α和β链的结合；以及iii)在结构水平上了解脂质结合的分子相互作用。为此，我们将使用一些生物物理方法来研究这种相互作用，包括磷脂双层和双胞环境中的多维固体和溶液核磁共振波谱，并结合体外和电子方法来研究单个α/β链和异二聚体α/β复合体的TMD-脂质相互作用。此外，我们将描述在二聚化和/或脂质结合中妥协的蛋白质变体。总之，这些研究将有助于我们理解TMD-TMD和TMD-脂质相互作用对MHC II类分子功能的贡献。

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.