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: 7968052
• 负责人: Robert E London
• 金额: $ 143.35万
• 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/7968052
• 关键词: Accounting; Acetylcholine; Address; Affinity; Allergens; Allergic; Amino Acids; Area; Arts; Bacteria; Base Excision Repairs; Base Pairing; Binding; Binding Proteins; Binding Sites; Biological Factors; Cells; Chemicals; Choline; Collaborations; Complex; Conflict (Psychology); DNA; DNA Binding; DNA Damage; DNA Polymerase beta; DNA Repair; DNA Structure; DNA ligase III; DNA-(apurinic or apyrimidinic site) lyase; Data; Dermatophagoides pteronyssinus antigen p 2; Dermatophagoides pteronyssinus antigen p 5; Dermatophagoides pteronyssinus antigen p 7; Environmental Health; Environmental Risk Factor; Enzymes; Exhibits; Exposure to; Genomic Instability; Genomics; Genotoxic Stress; Goals; Health; Homologous Gene; Human; Hydrogen Bonding; Immune; Individual; Isoleucine; Knowledge; Label; Length; Ligand Binding; Lipid Binding; Lipids; Literature; Marines; Mass Spectrum Analysis; Mediating; Methionine; Modeling; Modification; Molecular; Mollusca; N-terminal; NMR Spectroscopy; National Institute of Environmental Health Sciences; Natural Immunity; Neurotransmitters; Nicotine; Nicotinic Receptors; Nucleotide Excision Repair; Nucleotides; Organism; Pathway interactions; Patients; Play; Polynucleotide 5' -Hydroxyl-Kinase; Positioning Attribute; Process; Property; Proteins; Relative (related person); Repair Complex; Reporting; Research; Research Project Grants; Roentgen Rays; Role; Secondary to; Sequence Alignment; Single Strand Break Repair; Sodium Chloride; Solutions; Stress; Structure; System; Techniques; Thumb structure; Titrations; Triad Acrylic Resin; Tyrosine; Variant; Water; Work; X-Ray Crystallography; XRCC1 gene; alpha Bungarotoxin; base; gene correction; human XRCC1 protein; insight; interest; loris; methyl group; mimicry; mutant; protein expression; protein function; pyroglyphid; receptor; receptor binding; repair enzyme; repaired; research study; response; scaffold; stoichiometry

This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the area of environmental health. The primary emphasis during the recent review period involves several different research projects: 1) structural characterization of the repair complex formed from the XRCC1 N-terminal domain and DNA pol beta; 2) characterization of how the nucleotide excision repair protein UvrB interacts with various DNA structures; 3) characterization of a putative acetylcholine binding receptor from a marine annelid; 4) structural work on dust mite allergens. Project 1. The repair of damaged DNA involves multiple enzymatic steps, the activities of which must be spatially and temporally organized in order to optimize the repair process and to minimize the possibility of increased damage that could result from the release of repair intermediates such as nicked or gapped DNA. For the base excision repair (BER) and single strand break repair (SSBR) pathways, a major organizational role has been assigned to the protein XRCC1 acting as a scaffold for other base excision repair enzymes including DNA polymerase beta, DNA ligase III, PARP1 and 2, polynucleotide kinase (PNK) and AP endonuclease 1. Nevertheless, limited structural information corresponding to these putative repair complexes is currently available. We recently have utilized a combination of small angle X-ray scattering (SAXS), X-ray crystallography, and NMR spectroscopy to determine the structure of the complex formed from the XRCC1 N-terminal domain (XNTD) with DNA polymerase beta. The structure differs from several previously proposed models that involved simultaneous interactions with the Pol beta catalytic (palm) and nascent base pair (thumb) domains, as well as with the gapped DNA substrate, forming a protective "sandwich" around the damaged area. The present structural results indicate that Pol beta binds directly only with the thumb domain of Pol beta and is also not positioned to allow direct interactions with the gapped DNA repair intermediate. The interaction with Pol beta occurs primarily through the "V303 loop" and involves salt bridges, hydrophobic contacts, as well as multiple water-mediated hydrogen bonds. Areas of secondary structure agree well with the previously reported XRCC1 N-terminal domain structure, while there is substantial difference in many of the looop regions. Project 2. NMR characterization of the interaction of UvrB with hairpin DNA. UvrB, an essential component of the prokaryotic nucleotide excision repair (NER) pathway, plays a critical role in DNA damage recognition. Recent crystallographic structures of several UvrB-DNA complexes provide intriguing but incomplete insight into the damage recognition process. In order to further explore this interaction, we have performed NMR studies of the interaction of methyl-13Cmethionine UvrB from B. caldotenax with a compact hairpin DNA. The studies utilized a 14 nucleotide hairpin incorporating the highly stable seven nucleotide Hirao sequence and having a three nucleotide overhang at the 3'-terminus. Titration of the labeled UvrB with the hairpin perturbs the resonance of M350, which comes in direct contact with the DNA in a reported crystal structure, and also produces significant shifts for four methionine residues grouped together on helices F and G. The hypothesis that these perturbations are mediated primarily by the interaction of the 3'-nucleotide with F302 was confirmed by studies of the interaction of the DNA with the F302A mutant. In order to more specifically investigate the details of the UvrB conformational response, two additional methionine probes were introduced into the enzyme on the beta-hairpin (residues S91-N116) that has been proposed to play a central role in damage recognition. The I115M mutant, places a methionine residue at the base of the hairpin, while I109M places the methionine at the center of the hairpin. Based on the similarity of the response of the remaining methionine resonances to the addition of the DNA, it was concluded that the UvrB-DNA interaction was not significantly perturbed by either of these substitutions. Both the M109 and M115 resonances provided well resolved and sensitive probes for DNA binding. In particular, M115 exhibits a large upfield 1H shift that can be attributed to the altered position of the methionine methyl group relative to tyrosine residues Y92 and Y93. Tight binding of the DNA hairpin (KD < uM) is indicated by slow exchange of the methionine probe resonances. In several cases, shifts are observed at DNA:UvrB stoichiometries greater than 1:1, presumably due to secondary, weaker interactions of the DNA with additional portions of the DNA binding site. Project 3. The Yakel group (LN) has recently identified a homolog of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella capitata, from the annelid phylum. The Capitella capitata AChBP (cc-AChBP) has 21-30% amino acid identity with known molluscan AChBPs. Sequence alignments indicate that cc-AChBP has a shortened cys-loop compared to other cys-loop receptors, and a variation on a conserved C-loop triad, which is associated with ligand binding in other AChBPs and nicotinic acetylcholine receptor (nAChR) alpha subunits. Within the D-loop of cc-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand-binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 of cc-AChBP is glycosylated when expressed using HEK293 cells. Small angle X-ray scattering (SAXS) data are consistent with cc-AChBP existing as a soluble pentamer in solution. SAXS models for the apo-protein and nicotine-bound forms of the pentamer indicate large conformational changes of the protein upon binding of the ligand; these alterations appear not to be limited to just the C-loop/F-loop region. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to cc-AChBP with high affinity, with KD values of 28 microM, 200 nM, and 110 nM, respectively. Choline bound with a lower affinity (KD = 165 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may possess variation in hallmark motifs such as ligand-binding residues and cys-loop length, and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca Project 4. We have been investigating the structures of two allergens, Der p 5 and Der p 7, in order to provide insight into the basis for their allergenicity. These allergens generate an allergic response in 50% of patients, but the function of these proteins remains unknown. In collaboration with Lori Edwards in the protein expression core, and Lars Pedersen, a staff crystallographer, we have determined the structure of Der p 7, and have preliminary results on the structure of Der p 5. The initial results for the Der p 5 structure do not immediately provide clues related to its allergic effects, but it does appear to resolve a discrepancy in the literature regarding two conflicting structures of the homologous Blo t 5. The Der p 7 results, however, have very interesting implications. They appear to show that Der p 7 belongs to a class of proteins related to lipid binding in innate immunity, specifically in the TLR4 pathway. This is highly significant because of recent reports that describe another HDM allergen, Der p 2 can mimic MD2, also in the TLR4 pathway. The study proposed that allergen mimicry of innate immune proteins could account for allergenicity. Preliminary studies have demonstrated that Der p 7 does bind to lipid products from bacteria, in the predicted binding site.