We FRET so You Don’t Have To: New Models of the Lipoprotein Lipase Dimer

Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL–LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase.脂蛋白脂肪酶（Lipoprotein lipase, LPL）是一种负责清除血液中富含甘油三酯的脂蛋白的二聚体酶。尽管LPL在心血管健康中发挥关键作用，但其实验解析的三维结构至今尚未确定。这类结构将有助于理解导致患者罹患家族性LPL缺乏症的LPL突变，并助力靶向LPL的治疗策略开发。LPL结构研究的主要障碍在于，该蛋白稳定性较差，难以达到核磁共振或晶体学分析所需的表达量。本研究结合二硫键定位、计算建模以及单分子福斯特共振能量转移（single-molecule Förster resonance energy transfer, smFRET）所得数据，构建了更新后的LPL结构模型。我们通过优化活性LPL的成像条件、确定LPL上可连接荧光基团的位点，率先将smFRET技术应用于LPL研究。利用该方法，我们测定了LPL分子间的相互作用，以此生成实验约束条件，为LPL二聚体结构的新型计算模型提供依据。这些模型提示，LPL的二聚化界面可能与胰脂肪酶结构中由晶体堆积接触所暗示的二聚化界面存在差异。