Improved Hydroxyl Radical Footprinting for Modeling Protein Structure

描述(由申请人提供)：HIV疫苗开发的最受欢迎和最有希望的目标之一是来自gp120的免疫原，gp120是一种病毒进入CD4细胞所必需的包膜糖蛋白。最近，我们的合作者分离出了多种针对gp120的HIV广谱中和抗体，并表明它们需要某些N-连接的多糖才能发挥作用。然而，这些抗体中的一些并不结合游离的N-连接的多糖，这表明它们中的至少一些也可能与gp120的蛋白质部分相互作用。抗体.gp120复合物太大，无法通过核磁共振光谱产生高分辨率结构，而且由于糖蛋白似乎在抗体表位中发挥关键作用，因此复合物的结晶是一项艰巨的任务。计算建模是解决这一问题的一种有吸引力的方法，但纯粹的计算方法可能会生成精度有问题的模型，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-抗体复合体的羟基自由基蛋白质足迹来表征各种广谱中和抗体表位，这是一种基于对溶剂的可及性来标记各种氨基酸侧链的技术。为了提高足迹数据对准确、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进行精确定量以提高结构分辨率，以及通过归一化协议从足迹数据生成绝对溶剂可及表面积值。我们还建议开发一个适当的评分函数来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。通过这些改进及其在gp120-抗体复合体的表征中的应用，我们预计将产生准确的、实验受限的模型，正确地识别每种抗体的表位。这些模型对于合理设计免疫原，通过免疫在宿主体内产生相应的广中和抗体将是非常重要的。 与公共卫生相关：艾滋病毒仍然是世界各地的一种毁灭性疾病，特别是在发展中国家。尽管迫切需要，但艾滋病毒疫苗的开发仍然难以实现。最近，从HIV阳性患者中分离出一系列抗体，通过与包膜糖蛋白gp120相互作用，中和多种HIV血清型。这些抗体需要葡聚糖结合gp120，但至少其中一些抗体并不完全与葡聚糖结合。然而，抗体识别的表位尚未确定，因此不可能开发出通过疫苗在宿主中提高抗体的免疫原。我们建议利用羟基自由基足迹与计算建模相结合的方法来表征和建模gp120与每一种广泛中和抗体之间的复合体。这些模型将有助于合理开发免疫原，以研制出有效的抗HIV疫苗。

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.