Improved Hydroxyl Radical Footprinting for Modeling Protein Structure

描述（由申请人提供）：HIV疫苗开发中最受欢迎和最有希望的靶点之一是来自gp120的免疫原，gp120是病毒进入CD4细胞所必需的包膜糖蛋白。最近，我们的合作者已经分离出多种HIV广泛中和gp120的抗体，并表明它们需要某些n -链聚糖才能发挥活性。然而，其中一些抗体不结合游离的n链聚糖，这表明至少其中一些抗体也可能与gp120的蛋白质部分相互作用。抗体。gp120配合物太大，无法通过核磁共振光谱产生高分辨率结构，并且由于聚糖似乎在抗体表位中起关键作用，因此配合物的结晶是一项艰巨的任务。对于这个问题，计算建模是一种很有吸引力的方法，但是纯粹的计算方法可以生成精度可疑的模型，并且需要经验约束或测试才能生成可靠的模型。我们建议使用gp120抗体复合物的羟基自由基蛋白足迹来表征各种广泛中和的抗体表位，这是一种基于其对溶剂的可及性标记各种氨基酸侧链的技术。为了提高足迹数据对精确、高分辨率模型构建的有用性，我们建议开发一些改进的足迹方法，包括在氨基酸水平上精确定量以提高结构分辨率和标准化协议，以从足迹数据中生成绝对溶剂可达表面积值。我们还建议开发一个适当的评分函数，以利用溶剂可达表面积作为分子动力学模拟中的约束，类似于使用距离约束。从这些改进及其在gp120抗体复合物表征中的应用来看，我们期望产生准确的，实验约束的模型，正确识别每个抗体的表位。这些模型对合理设计免疫原，通过免疫提高宿主体内相应的广泛中和抗体具有重要意义。

DESCRIPTION (provided by applicant): One of the most popular and promising targets for HIV vaccine development are immunogens derived from gp120, an envelope glycoprotein essential for viral entry into CD4 cells. Recently, our collaborators have isolated multiple HIV broadly neutralizing antibodies to gp120 and shown that they require certain N-linked glycans for activity. However, some of these antibodies do not bind the free N-linked glycans, suggesting that at least some of them may also interact with the protein portion of gp120. The antibody.gp120 complexes too large for generation of high-resolution structures by NMR spectroscopy, and as the glycan appear to play key roles in the antibody epitopes, crystallization of the complexes is a daunting task. Computational modeling is an attractive approach for this problem, but purely computational approaches can generate models of questionable accuracy, and require empirical constraints or testing in order to generate a reliable model. We propose to characterize the various broadly neutralizing antibody epitopes using hydroxyl radical protein footprinting of the gp120-antibody complexes, a technique that labels a broad variety of amino acid side chains based on their accessibility to solvent. In order to improve the usefulness of the footprinting data for accurate, high-resolution model building, we propose to develop a number of improved footprinting methods, including accurate quantitation at the amino acid level to improve structural resolution and normalization protocols to generate absolute solvent accessible surface area values from footprinting data. We also propose to develop an appropriate scoring function to utilize solvent accessible surface areas as a constraint in molecular dynamics simulations, analogous to the use of distance constraints. From these improvements and their application to the characterization of gp120-antibody complexes, we anticipate the generation of accurate, experimentally-constrained models that correctly identify the epitope for each antibody. These models will be very important for the rational design of immunogens to raise the corresponding broadly neutralizing antibodies in a host through immunization. PUBLIC HEALTH RELEVANCE: HIV remains a devastating disease throughout the world, especially in developing countries. Despite the urgent need, development of a vaccine to HIV remains elusive. Recently, a series of antibodies that neutralize a broad variety of HIV serotypes through interaction with the envelope glycoprotein gp120 have been isolated from HIV-positive patients. These antibodies require the glycan to bind gp120, but at least some of them do not bind solely to the glycan. However, the epitope that the antibody recognizes has not been defined, so it is not possible to develop an immunogen to raise the antibody in a host through a vaccine. We propose to utilize hydroxyl radical footprinting coupled with computational modeling to characterize and model the complex between gp120 and each of the broadly neutralizing antibodies. These models will aid in the rational development of immunogens to formulate effective anti-HIV vaccines.