Nmr Studies Of Biomolecular Structure, Function, And Dynamics

生物分子结构、功能和动力学的核磁共振研究

• 批准号: 8336566
• 负责人: Robert E London
• 金额: $ 83.7万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/8336566
• 关键词: Active Sites; Adopted; Affinity; B-DNA; BRCT Domain; Base Excision Repairs; Base Pairing; Binding; Biochemical; Biological Factors; C-terminal; Chemicals; Circular Dichroism; Complement 3a; Complex; Coupling; Cytidine; DNA; DNA Damage; DNA Polymerase beta; DNA Repair; DNA Structure; Data; Deoxycytidine; Dimerization; Electrostatics; Environmental Risk Factor; Enzymes; Evaluation; Event; Exposure to; Genomic Instability; Genomics; Guanidines; Health; Human; Hydrogen Bonding; Hydroxylation; Individual; Ions; Knowledge; Label; Lead; Ligase; Lysine; Measurement; Mediating; Membrane Proteins; Metals; Methods; Modeling; Modification; Molecular Conformation; Molecular Models; Motion; Mutation; N-terminal; NMR Spectroscopy; Nucleic Acids; Nucleotides; Physiological; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymerase; Positioning Attribute; Process; Proteins; Publications; Purines; Pyrimidine; Repair Complex; Resolution; Ribonucleotides; Role; Scaffolding Protein; Series; Solutions; Stress; Structure; Vertebral column; Work; X-Ray Cross Complementing Group; XRCC1 gene; Zinc; adverse outcome; assault; base; cytotoxic; density; disulfide bond; divalent metal; enzyme structure; human XRCC1 protein; insight; man; melting; molecular modeling; protein complex; purine; repair enzyme; repaired; response

This project utilizes state-of-the-art NMR spectroscopy to characterize DNA damage and to determine the structure of enzymes involved in base excision repair and the intact base excision repair complex. The primary emphasis during the recent review period includes 1) structural characterization of the repair complex formed from the XRCC1 N-terminal domain and DNA pol beta; 2) characterization of the interaction of XRCC1 BRCT domains with cognate BRCT domains on Ligase IIIa and on Poly(ADP-ribose) polymerase-1 (PARP-1); 3) characterization of the structure of DNA containing isolated ribonucleotides; 4) understanding the relationship of conformational activation of Pol beta to polymerase fidelity Project 1. Base excision repair (BER) is a complex process requiring the coordinated activities of multiple enzymes that interpret the damage, excise the damaged components, and resynthesize the DNA. Current understanding of the BER repair complex involves formation of transient complexes with the damaged DNA. These repair complexes are organized by the scaffold protein, XRCC1. However, the complex formed between the C-terminal BRCT domains of XRCC1 and Ligase IIIalpha is constitutive. Although the Ligase IIIalpha was the first XRCC1 binding partner to be identified, characterization of the underlying complex has eluded structural determination. Furthermore, all existing data for BRCT domain-mediated protein-protein complexes involved pairs of tandem BRCT domains, while this is not the case for the XRCC1-LigaseIIIalpha complex. In the absence of structural data, alternative proposals for this interaction have been advanced. Interpretation of these models is complicated by the formation of homodimers which, depending on the model, may either contribute to, or compete with heterodimer formation. During the past year, we determined the structures of both the X1BRCTb and the L3BRCT homodimers, as well as the heterodimeric X1BRCTb:L3BRCT complex. The initial structural characterization of the heterodimer suggested that additional residues might be involved in complex formation. On this basis, we studied a longer X1BRCTb construct, containing residues comprising the interdomain linker region immediately preceding X1BRCTb. Structural characterization of this extended construct revealed a structural motif which leads to significant stabilization of the heterodimer and dictates heterodimer/homodimer selectivity. The role of the additional linker residues was further confirmed by melting studies performed using circular dichroism. These data provide important fundamental insights into the structural basis of BRCT-mediated dimerization events. Additionally, the data resolve questions related to the organization of this critically important DNA repair complex. Project 2. We have also continued our studies of the interaction of the N-terminal domain of XRCC1 with the repair polymerase, Pol beta. We previously had determined that the N-terminal domain of XRCC1, X1NTD, is able to adopt two dramatically different structures, related to the presence or absence of a disulfide bond. In order to better understand the physiological role of this reversible disulfide bond formation, we have worked to better define the conditions that lead to the oxidized form of X1NTD. Our previous study had identified a mutation, I4D, that helped to stabilize the oxidized form. More recent studies have identified other experimental conditions that also stabilize this form of the protein. Based on the analysis of the backbone shifts using TALOS, we have been able to demonstrate that the solution structure obtained under these conditions is similar to that observed in the crystalline complex with Pol beta. Nevertheless, in order to understand the physiological significance of the oxidized form of X1NTD, it is necessary to further stabilize this form. We are currently exploring various mutational strategies that may provide a more succeessful degree of stabilization than the initial mutation used in our first study, without altering the electrostatic surface of the protein as significantly as the I4D substitution. Project 3. We have continued our evaluation of the effects of metals on DNA pol beta by studying the conformational response to divalent zinc. Binding of the catalytic divalent ion to the ternary DNA pol beta/gapped DNA-dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg2+ and Zn2+ on the conformational activation process based on NMR measurements of methyl 13Cmethionine-labeled DNA polymerase beta. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/one-nucleotide gapped DNA complex in the absence of a complementary incoming nucleotide. This conformational activation can be abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results suggest that metal-binding is able to promote translocation of the primer terminus base pair into the active site, expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket, and motions of polymerase subdomains that close the active site. These results have important implications for fidelity and pyrophosphorolysis. Project 4. This project is directed at characterizing the interaction of the first XRCC1 BRCT domain, X1BRCTa, with its previously identified binding partner, PARP1. Although previous publications had indicated that X1BRCTa binds to the BRCT domain of PARP1, we find that, after an extensive series of studies, there is no significant binding affinity between the two BRCT domains. One possible resolution of this inconsistency is that the presence of poly(ADP-ribose) modification of the residues adjacent to the PARP1 BRCT domain is essential to obtain binding. We are currently evaluating this possibility. Project 5. Effect of 2'-hydroxylation on DNA structure. It has recently been demonstrated that the extent of incorporation of ribonucleotides by replicative polymerases is sufficient to make 2'-hydroxylation the most common mutation in newly synthesized DNA. We have used the Dickerson dodecamer as a model for the analysis of the effect of an isolated ribonucleotide on DNA structure. The self-complementary sequence investigated, d(CGC)rGd(TTAAGCGC), contains two symmetrically positioned guanidine residues paired with deoxycytidine residues. Consistent with previous studies of mixed ribo/deoxyribo nucleic acids, the absence of an observable H1'-H2' scalar coupling interaction is indicative of a C3'-endo conformation for the cytidine residue. However, longer range structural perturbations resulting from the presence of the ribonucleotide appear to be quite modest, so that the B-form DNA structure is maintained. During the past year we performed a more thorough analysis of complete structure of our model nucleotide, and also performed extensive molecular modeling calculations in order to more fully evaluate these results, and to extrapolate the conclusions to DNA containing a higher density of ribonucleotide substitutions.

该项目利用最先进的NMR光谱来表征DNA损伤，并确定涉及基础切除修复和完整碱基切除修复复合物的酶的结构。最近的审查期间的主要重点包括1）由XRCC1 N末端结构域和DNA POL Beta形成的修复复合物的结构表征； 2）表征XRCC1 BRCT结构域与连接酶IIIA和聚（ADP-核糖）聚合酶1（PARP-1）上的同源BRCT结构域的相互作用； 3）表征含有分离的核糖核苷酸的DNA结构； 4）了解POLβ与聚合酶保真度的构象激活的关系 项目1。基本切除修复（BER）是一个复杂的过程，需要多种酶的协调活动，这些酶解释损坏，切除受损的组件并重新合成DNA。当前对BER修复复合物的理解涉及与受损的DNA形成瞬态复合物。 这些修复复合物由支架蛋白XRCC1组织。 但是，XRCC1的C末端BRCT结构域与连接酶IIIALPHA之间形成的复合物是组成型。 尽管连接酶IIIALPHA是要鉴定的第一个XRCC1结合伴侣，但基础复合物的表征已经取决于结构性测定。 此外，BRCT结构域介导的蛋白质蛋白质复合物的所有现有数据都涉及成对的串联BRCT结构域，而XRCC1-ligaseiiialpha复合物并非如此。在没有结构数据的情况下，已经提出了有关这种相互作用的替代建议。 这些模型的解释是由于同二聚体的形成而复杂的，这些二聚体的形成取决于模型，可能有助于或与异二聚体形成竞争或竞争。在过去的一年中，我们确定了X1BRCTB和L3BRCT同型二聚体以及异二聚体X1BRCTB：L3BRCT复合物的结构。 异二聚体的初始结构表征表明，其他残基可能参与复杂形成。 在此基础上，我们研究了一个更长的X1BRCTB构建体，其中包含包含在X1BRCTB之前的域间接头区域的残基。 该扩展构建体的结构表征揭示了一个结构基序，从而导致异二聚体的显着稳定，并决定异二聚体/同型二聚体选择性。 通过使用圆形二色性研究进行的融化研究进一步证实了其他接头残基的作用。 这些数据为BRCT介导的二聚事件的结构基础提供了重要的基本见解。 此外，数据解决了与此至关重要的DNA修复复合物的组织有关的问题。 项目2。我们还继续研究XRCC1与修复聚合酶POL Beta的N末端结构域相互作用。 我们先前已经确定XRCC1 X1NTD的N末端结构域能够采用与存在或不存在二硫键有关的两个急剧不同的结构。为了更好地理解这种可逆的二硫键形成的生理作用，我们努力更好地定义导致X1NTD氧化形式的条件。 我们先前的研究确定了一个突变I4D，有助于稳定氧化形式。 最近的研究已经确定了其他实验条件，这些实验条件也稳定了这种形式的蛋白质。基于使用TALOS对主链移动的分析，我们能够证明在这些条件下获得的溶液结构类似于与POL Beta的晶体复合物中观察到的溶液结构。 然而，为了了解X1NTD氧化形式的生理意义，有必要进一步稳定这种形式。目前，我们正在探索各种突变策略，这些突变策略可能比首次研究中使用的初始突变提供了更成功程度的稳定度，而没有像I4D取代那样明显改变蛋白质的静电表面。 项目3。我们通过研究对二价锌的构象反应，继续评估金属对DNA POL Beta的影响。 催化二价离子与三元DNA POLβ/间隙DNA-DNTP复合物的结合被认为代表了催化复合物组装的最后一步，因此是复制性保真度的关键决定因素。我们已经分析了Mg2+和Zn2+对基于甲基13cmethion氨基标记的DNA聚合酶β的NMR测量值的构象激活过程的影响。出乎意料的是，两种二价金属能够在没有互补的输入核苷酸的情况下产生聚合酶/一核苷酸间隙DNA复合物的模板依赖性构象激活。可以通过用赖氨酸代替GLU295来消除这种构象活化，从而中断稳定封闭构象所需的关键氢键。这些和其他结果表明，金属结合能够促进底漆末端对的易位进入活性位点，从模板结合口袋中驱逐了未配对的嘧啶，而不是嘌呤，以及关闭活性位点的聚合酶子域的运动。这些结果对忠诚度和焦磷酸解液具有重要意义。 项目4。该项目旨在表征第一个XRCC1 BRCT域X1BRCTA的相互作用，并及其先前确定的绑定伙伴PARP1。 尽管以前的出版物表明X1BRCTA与PARP1的BRCT结构域结合，但我们发现，经过一系列广泛的研究，两个BRCT域之间没有显着的结合亲和力。 这种不一致的一种可能的分辨率是，与PARP1 BRCT域相邻的残基进行聚（ADP-核糖）的存在对于获得结合至关重要。 我们目前正在评估这种可能性。 项目5。2'-羟基化对DNA结构的影响。最近已经证明，通过复制聚合酶掺入核糖核苷酸的程度足以使2'-羟基化成为新合成的DNA中最常见的突变。我们已经使用迪克森十二焦点作为分析分离的核糖核苷酸对DNA结构的影响的模型。研究的自我平衡序列D（CGC）RGD（TTAAGCGC）包含两个对称定位的鸟根残基，与脱氧胞苷残基配对。与以前对混合核/脱氧核酸核酸的研究一致，缺乏可观察到的H1'-H2'标量偶联相互作用，这表明了胞苷残基的C3'-末代构象。但是，由于存在核糖核苷酸的存在而产生的较长范围的结构扰动似乎很小，因此可以维持B形DNA结构。在过去的一年中，我们对模型核苷酸的完整结构进行了更彻底的分析，并进行了广泛的分子建模计算，以便更全面地评估这些结果，并将结论推断为含有较高核糖核苷酸替代密度的DNA。