Structural Characterization Of Proteins

蛋白质的结构表征

• 批准号: 7964352
• 负责人: PETER D. SUN
• 金额: $ 101.35万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964352
• 关键词: Activated Natural Killer Cell; Adopted; Amino Acids; Binding; Binding Sites; Biological Assay; Buffers; CXCL10 gene; CXCL9 gene; Cell-Mediated Cytolysis; Cells; Cloning; Complex; Crystallization; Cysteine Protease; Cytolysis; Employee Strikes; Equilibrium; Glutamine; HLA-Cw3; Immune system; Immunoglobulin G; Interferon Type II; KLRD1 gene; LGLA; Ligand Binding; Ligands; Link; Macrophage-1 Antigen; Mass Spectrum Analysis; Mediating; Methods; Molecular Profiling; Muramidase; Mutation; Myeloid Cells; NK Cell Activation; Natural Killer Cells; Peptides; Proteins; Reaction; Reporter; Research Personnel; Screening procedure; Solutions; Streptococcus pyogenes; Structure; Surface Antigens; Tumor Cell Line; Viral; Virulence Factors; X-Ray Crystallography; chemokine; cytotoxicity; design; dimer; improved; interest; killer inhibitory receptor; knowledge base; meetings; natural killer cell protein 44-kDa; protein function; protein structure; receptor; research study; structural biology; tumor

The function of natural killer (NK) and myeloid cells depends on the balance of a number of activating and inhibitory receptors. Natural cytotoxicity receptors (NCR) are the major receptors responsible for NK cell-mediated lysis of tumor and viral-infected cells. Recent cloning of three NCRs, NKp46, NKp44, and NKp30, prompted studies by a number of investigators to elucidate their function as well as to identify their ligands. In the past few years, we have characterized the structure and function of several inhibitory as well as activating NK cell receptors. These include the crystal structures of CD94, KIR2DL2 and its complex with HLA-Cw3, NKG2D and its complex with ULBP3, NKp46 and TREM-1. Despite the progress made in understanding the function of NK receptors in the past few years, the ligands recognized by NCR remain unknown. Our effort of understanding the function of NCR has led a structural solution for NKp46. The structure revealed striking resemblence between NKp46 and KIR receptors and enabled us to propose a potential ligand binding site. To search for the potential ligands of NCR, we designed a high throughput binding assay that enables us to systematically evaluate the binding of NCR to known cell surface antigens. In an effort to understand the mechanism of a virulence factor, mac-1, from Group A Streptococcus, we have revealed that the protein functions as a cysteine protease specific for IgG. The structure shows that it adopts a cysteine protease fold with a unique dimer formation and that the mutations at the dimer interface drastically reduced the catalytic activity. Through solution binding experiments, we identified several chemokines, in particular the interferon gamma inducible chemokines CXCL9, CXCL10 as potential ligands of NCR. We are currently evaluating their functional relevance in NCR mediated cellular cytotoxicity. We have screened a number of tumor cell lines for their expression profiles of these chemokines and are constructing NCR reporter cells to evaluate the ability of chemokines to functionally activate the activating NK receptors. Heavy atom derivatization is routinely used in protein structure determination and is thus of critical importance in structural biology. In order to replace the current trial-and-error heavy atom derivative screening with a knowledge-based rational derivative selection method, we systematically examined the reactivity of more than forty heavy atom compounds over a wide range of buffer and pH on peptides which contained a single reactive amino acid residue. Met, Cys and His-containing peptides were derivatized against Hg, Au and Pt compounds while Tyr, Glu, Asp, Asn, and Gln-containing peptides were assessed against Pb compounds. A total of 1668 reactive conditions were examined using mass spectrometry and compiled into heavy atom reactivity tables. The results showed that heavy atom derivatization reactions are highly linked to buffer and pH with the most accommodating buffer being MES at pH 6. A group of 21 compounds were identified as most successful irrespective of ligand or buffer/pH conditions. To assess the applicability of the peptide heavy atom reactivity to proteins, lysozyme crystals were derivatized with a list of peptide reactive compounds that included both known and new compounds for lysozyme derivatization. The results showed highly consistent heavy atom reactivities between the peptide and lysozyme.

天然杀伤（NK）和髓样细胞的功能取决于许多激活和抑制受体的平衡。天然细胞毒性受体（NCR）是负责NK细胞介导的肿瘤和病毒感染细胞裂解的主要受体。 最近对三个NCRS，NKP46，NKP44和NKP30的克隆促使许多研究者的研究促进了其功能以及识别其配体的功能。在过去的几年中，我们表征了几种抑制性以及激活NK细胞受体的结构和功能。 其中包括CD94，KIR2DL2的晶体结构及其与HLA-CW3，NKG2D的复合物以及与ULBP3，NKP46和TREM-1的复合物。 尽管在过去几年中了解NK受体的功能方面取得了进展，但NCR认可的配体仍然未知。 我们理解NCR功能的努力为NKP46提供了结构解决方案。 该结构揭示了NKP46和KIR受体之间的惊人相似之处，使我们能够提出潜在的配体结合位点。 为了搜索NCR的潜在配体，我们设计了一种高通量结合测定法，使我们能够系统地评估NCR与已知细胞表面抗原的结合。 为了了解A组链球菌的毒力因子Mac-1的机理，我们揭示了该蛋白质是对IgG特异的半胱氨酸蛋白酶的作用。 该结构表明，它采用具有独特二聚体形成的半胱氨酸蛋白酶褶皱，并且二聚体界面处的突变大大降低了催化活性。 通过溶液结合实验，我们确定了几种趋化因子，特别是干扰素γ-诱导型趋化因子CXCL9，CXCL10是NCR的潜在配体。 我们目前正在评估它们在NCR介导的细胞细胞毒性中的功能相关性。 我们已经筛选了许多肿瘤细胞系的这些趋化因子的表达谱，并正在构建NCR报告基细胞以评估趋化因子在功能上激活活化的NK受体的能力。 重原子衍生化通常用于蛋白质结构测定中，因此在结构生物学中至关重要。为了用基于知识的有理衍生物选择方法代替当前的反复试验性原子衍生物筛选，我们系统地检查了在包含单个反应性氨基酸残基的肽上，在肽上广泛的缓冲液和pH值中的40多种重原子化合物的反应性。 MET，CYS和含有他的含有Hg，Au和PT化合物的肽被衍生而成，而Tyr，Glu，ASP，ASN和含GLN的肽对PB化合物进行了评估。使用质谱法检查了总共1668个反应性条件，并将其编译成沉重的原子反应表。结果表明，重原子衍生反应与缓冲液和pH值高度相关，最容易容纳的缓冲液是pH 6的MES。无论配体或缓冲液/pH条件如何，一组21种化合物被确定为最成功的。 为了评估肽沉重原子对蛋白质的适用性，用肽反应性化合物列表衍生溶菌酶晶体，其中包括已知和新化合物的溶菌酶衍生化化合物。 结果显示肽和溶菌酶之间高度一致的重原子反应率。