Structure and dynamics of bacterial membrane protein - receptor interactions

细菌膜蛋白-受体相互作用的结构和动力学

• 批准号: 8888106
• 负责人: Linda M Columbus
• 金额: $ 31.22万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8888106
• 关键词: Affinity; Alleles; Antibiotic Resistance; Bacteria; Binding; Biological; Biological Assay; Biological Testing; CEACAM1; Carcinoembryonic Antigen; Cell Adhesion; Cell Adhesion Molecules; Cells; Chimera organism; Complex; Consensus; Data; Disease; Electron Spin Resonance Spectroscopy; Event; Exhibits; Family; Fluorescence Polarization; Funding; Genetic Recombination; Glean; Goals; Hela Cells; Heparan Sulfate Proteoglycan; Human; Hybrids; In Situ; In Vitro; Knowledge; Life; Liposomes; Measures; Mediating; Membrane; Membrane Proteins; Meningitis; Methods; Molecular; Molecular Conformation; Neisseria; Neisseria gonorrhoeae; Nuclear Magnetic Resonance; Pathogenesis; Pathway interactions; Peptides; Phagocytes; Play; Positioning Attribute; Proline; Property; Protein Binding; Proteins; Research; Resolution; Risk; Sampling; Sequence Alignment; Solutions; Specificity; Structure; System; Testing; Therapeutic; Time; Variant; X-Ray Crystallography; base; conformer; design; driving force; extracellular; in vivo; insight; interest; molecular dynamics; molecular recognition; mutant; nanosecond; neutrophil; novel; protein complex; public health relevance; receptor; receptor binding; structural biology; three dimensional structure

 DESCRIPTION (provided by applicant): Opa proteins from Neisseria gonorrhoeae and N. meningitidis pose an intriguing biological riddle: How can a structure that is tolerant of sequence diversity bind to the same family of receptors, and also provide receptor selectivity among the receptor variants? Opa is a class of outer membrane proteins that bind to various host receptors that induce engulfment of the bacterium. The Opa receptor specificity is predominantly determined by two segments (hypervariable, HV, 1 and 2) in extracellular loops 2 and 3. The Opa barrel sequences exhibit 75% identity, but these loop regions display high sequence diversity. A structural motif for receptor engagement and selectivity has not been identified. Our overarching goal is to determine how Opa proteins interact with their cognate human receptors in order to decipher the mechanism of the molecular recognition and the specific molecular determinants of several Opa-receptor interactions. Knowledge of the structure, dynamics, and specific interactions of Opa proteins and receptors will be used to design targeted liposome delivery to human cells. We have only begun to unravel the answers to this complex puzzle. Our recent Opa60 structure and dynamics data indicate the extracellular loops are disordered, yet sample a restricted volume such that frequent short-lived transient interactions occur between the loops on the nanosecond timescale. These results partly answer the riddle - the structure is not discrete or well-defined, but rather can tolerate sequence variability in the HV sequences as a mechanism for binding. Molecular dynamics simulations indicate the loops engage in transient interactions, specifically between the HV1 and HV2 regions; this may be a mechanism by which the protein can sample conformers in which HV1 and HV2 are in proximity and can bind receptor. Though this protein system may be inherently ill-defined in terms of the typical structural biology paradigm, we speculate that a specific, well-defined mechanism is at play. We propose a hypothesis wherein the clustering of hydrophobic residues mediates transient interactions between HV1 and HV2. Once localized through these interactions, we hypothesize further that two or more states are sampled on the µs - ms time scale with a sub-set of conformations competent for receptor binding. To test these hypotheses, we will use a hybrid method approach to determine the dynamics and interactions of Opa60 in the unbound and receptor bound states. In addition, we have developed in vivo assays that tests the biological significance of the Opa- receptor molecular determinants that we elucidate with in vitro studies. The combined results of the proposed studies will determine how Opa proteins interact with their cognate human receptors providing insights into the pathogenesis and potential treatments for N. gonorrhoeae and N. meningitides, of which the former has reached "super bug" status because of antibiotic resistance. Longer term, this knowledge will serve as a platform for designing a delivery system that targets foreign entities into specific subsets of human cells.

 描述（应用程序提供）：来自淋病奈瑟氏菌的OPA蛋白质和脑膜炎N. 多样性与同一受体家族结合，还提供受体变体之间的受体选择性？ OPA是一类外膜蛋白，与影响细菌吞噬的各种宿主受体结合。 OPA受体的特异性主要由细胞外环2和3中的两个段（高变量，HV，1和2）确定。OPA枪管序列暴露了75％的身份，但这些环路区域显示高序列多样性。尚未确定用于受体参与和选择性的结构基序。我们的总体目标是确定OPA蛋白如何与其同源人体受体相互作用，以破译分子识别的机制以及几种OPA受体相互作用的特定分子决定剂。对OPA蛋白和受体的结构，动力学和特定相互作用的了解将用于设计靶向脂质体向人类细胞的递送。我们刚刚开始解开这个复杂难题的答案。我们最近的OPA60结构和动力学数据表明细胞外回路是无序的，但是样品限制了体积，因此经常短暂的短暂瞬态相互作用发生在纳米秒时间上的环之间。这些结果部分回答了谜语 - 结构不是离散的或明确的，而是可以忍受HV序列中的序列可变性，作为结合的机制。分子动力学模拟表明，循环参与了瞬态相互作用，特别是在HV1和HV2区域之间。这可能是一种机制，蛋白质可以采样HV1和HV2处于接近性并可以结合受体的构象异构体。尽管这种蛋白质系统可能在典型的结构生物学范式方面固有地定义不明，但我们推测一种特定的，定义明确的机制正在发挥作用。我们提出了一个假设，其中疏水残差的聚类介导了HV1和HV2之间的短暂相互作用。一旦通过这些相互作用进行了定位，我们进一步假设在µS -MS时间尺度上采样了两个或多个状态，并具有符合受体结合的构象的子集。为了检验这些假设，我们将使用一种混合方法来确定在未结合和受体结合态中OPA60的动力学和相互作用。此外，我们开发了体内测定，该测定测试了我们通过体外研究阐明的透明质受体分子确定剂的生物识别意义。拟议研究的综合结果将确定Opa蛋白如何与其同源人体相互作用，从而为淋病猪笼草和脑膜炎猪笼草提供了有关发病机理和潜在处理的见解，而前者由于抗生素耐药性而达到了“超级虫”状态。从长远来看，这些知识将作为设计将外国实体靶向特定人类细胞子集的输送系统的平台。