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author:("Zhang, deqing")
1.  Changes in Forest Soil Properties in Different Successional Stages in Lower Tropical China 
PLoS ONE  2013;8(11):e81359.
Background
Natural forest succession often affects soil physical and chemical properties. Selected physical and chemical soil properties were studied in an old-growth forest across a forest successional series in Dinghushan Nature Reserve, Southern China.
Methodology/Principal Findings
The aim was to assess the effects of forest succession change on soil properties. Soil samples (0–20 cm depth) were collected from three forest types at different succession stages, namely pine (Pinus massoniana) forest (PMF), mixed pine and broadleaf forest (PBMF) and monsoon evergreen broadleaf forest (MEBF), representing early, middle and advanced successional stages respectively. The soil samples were analyzed for soil water storage (SWS), soil organic matter (SOM), soil microbial biomass carbon (SMBC), pH, NH4+-N, available potassium (K), available phosphorus (P) and microelements (available copper (Cu), available zinc (Zn), available iron (Fe) and available boron (B)) between 1999 and 2009. The results showed that SWS, SOM, SMBC, Cu, Zn, Fe and B concentrations were higher in the advanced successional stage (MEBF stage). Conversely, P and pH were lower in the MEBF but higher in the PMF (early successional stage). pH, NH4+-N, P and K declined while SOM, Zn, Cu, Fe and B increased with increasing forest age. Soil pH was lower than 4.5 in the three forest types, indicating that the surface soil was acidic, a stable trend in Dinghushan.
Conclusion/Significance
These findings demonstrated significant impacts of natural succession in an old-growth forest on the surface soil nutrient properties and organic matter. Changes in soil properties along the forest succession gradient may be a useful index for evaluating the successional stages of the subtropical forests. We caution that our inferences are drawn from a pseudo-replicated chronosequence, as true replicates were difficult to find. Further studies are needed to draw rigorous conclusions regarding on nutrient dynamics in different successional stages of forest.
doi:10.1371/journal.pone.0081359
PMCID: PMC3828269  PMID: 24244738
2.  Allelic Variation in a Cellulose Synthase Gene (PtoCesA4) Associated with Growth and Wood Properties in Populus tomentosa 
G3: Genes|Genomes|Genetics  2013;3(11):2069-2084.
Lignocellulosic biomass from trees provides a renewable feedstock for biofuels, lumber, pulp, paper, and other uses. Dissecting the mechanism underlying natural variation of the complex traits controlling growth and lignocellulose biosynthesis in trees can enable marker-assisted breeding to improve wood quality and yield. Here, we combined linkage disequilibrium (LD)-based association analysis with traditional linkage analysis to detect the genetic effect of a Populus tomentosa cellulose synthase gene, PtoCesA4. PtoCesA4 is strongly expressed in developing xylem and leaves. Nucleotide diversity and LD in PtoCesA4, sampled from the P. tomentosa natural distribution, revealed that PtoCesA4 harbors high single nucleotide polymorphism (SNP) diversity (πT = 0.0080 and θw = 0.0098) and low LD (r2 ≥ 0.1, within 1400 bp), demonstrating that the potential of a candidate-gene-based LD approach in understanding the molecular basis underlying quantitative variation in this species. By combining single SNP, multi-SNP, and haplotype-based associations in an association population of 460 individuals with single SNP linkage analysis in a family-based linkage populations (1200 individuals), we identified three strong associations (false discovery rate Q < 0.05) in both populations. These include two nonsynonymous markers (SNP49 associated with α-cellulose content and SNP59 associated with fiber width) and a noncoding marker (SNP18 associated with α-cellulose content). Variation in RNA transcript abundance among genotypic classes of SNP49 was confirmed in these two populations. Therefore, combining different methods allowed us to examine functional PtoCesA4 allelic variation underlying natural variation in complex quantitative traits related to growth and lignocellulosic biosynthesis.
doi:10.1534/g3.113.007724
PMCID: PMC3815066  PMID: 24048648
linkage disequilibrium; linkage analysis; multi-locus association models; Populus tomentosa; RNA transcript analysis; single nucleotide polymorphism
3.  Variation in Genomic Methylation in Natural Populations of Chinese White Poplar 
PLoS ONE  2013;8(5):e63977.
Background
It is thought that methylcytosine can be inherited through meiosis and mitosis, and that epigenetic variation may be under genetic control or correlation may be caused by neutral drift. However, DNA methylation also varies with tissue, developmental stage, and environmental factors. Eliminating these factors, we analyzed the levels and patterns, diversity and structure of genomic methylcytosine in the xylem of nine natural populations of Chinese white poplar.
Principal Findings
On average, the relative total methylation and non-methylation levels were approximately 26.567% and 42.708% (P<0.001), respectively. Also, the relative CNG methylation level was higher than the relative CG methylation level. The relative methylation/non-methylation levels were significantly different among the nine natural populations. Epigenetic diversity ranged from 0.811 (Gansu) to 1.211 (Shaanxi), and the coefficients of epigenetic differentiation (GST = 0.159) were assessed by Shannon’s diversity index. Co-inertia analysis indicated that methylation-sensitive polymorphism (MSP) and genomic methylation pattern (CG-CNG) profiles gave similar distributions. Using a between-group eigen analysis, we found that the Hebei and Shanxi populations were independent of each other, but the Henan population intersected with the other populations, to some degree.
Conclusions
Genome methylation in Populus tomentosa presented tissue-specific characteristics and the relative 5′-CCGG methylation level was higher in xylem than in leaves. Meanwhile, the genome methylation in the xylem shows great epigenetic variation and could be fixed and inherited though mitosis. Compared to genetic structure, data suggest that epigenetic and genetic variation do not completely match.
doi:10.1371/journal.pone.0063977
PMCID: PMC3660595  PMID: 23704963
4.  Sexual Dimorphism Floral MicroRNA Profiling and Target Gene Expression in Andromonoecious Poplar (Populus tomentosa) 
PLoS ONE  2013;8(5):e62681.
Although the molecular basis of poplar sex-specific flower development remains largely unknown, increasing evidence indicates an essential role for microRNAs (miRNAs). The specific miRNA types and precise miRNA expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. This system, combined with high-throughput sequencing and computational analysis, allowed us to characterize sex-specific miRNAomes from female and male flowers. Comparative miRNAome analysis combined with quantitative real-time PCR revealed the expression patterns of 27 miRNAs in poplar flower and showed that the targets of these miRNAs are involved in flower organogenesis, Ca2+ transport, phytohormone synthesis and metabolism, and DNA methylation. This paper describes a complex regulatory network consisting of these miRNAs expressed in sex-specific flower development in a dioecious plant. The conserved and novel miRNA locations were annotated in the Populus trichocarpa genome. Among these, miRNA Pto-F70 and 4 targets are located in the sex-determination regions of chromosome XIX. Furthermore, two novel miRNAs, Pto-F47 and Pto-F68, were shown for the first time to be regulatory factors in phytohormone interactions. To our knowledge, this report is the first systematic investigation of sex-specific flower-related miRNAs and their targets in poplar, and it deepens our understanding of the important regulatory functions of miRNAs in female and male flower development in this dioecious plant.
doi:10.1371/journal.pone.0062681
PMCID: PMC3646847  PMID: 23667507
5.  Response of Soil Respiration to Acid Rain in Forests of Different Maturity in Southern China 
PLoS ONE  2013;8(4):e62207.
The response of soil respiration to acid rain in forests, especially in forests of different maturity, is poorly understood in southern China despite the fact that acid rain has become a serious environmental threat in this region in recent years. Here, we investigated this issue in three subtropical forests of different maturity [i.e. a young pine forest (PF), a transitional mixed conifer and broadleaf forest (MF) and an old-growth broadleaved forest (BF)] in southern China. Soil respiration was measured over two years under four simulated acid rain (SAR) treatments (CK, the local lake water, pH 4.5; T1, water pH 4.0; T2, water pH 3.5; and T3, water pH 3.0). Results indicated that SAR did not significantly affect soil respiration in the PF, whereas it significantly reduced soil respiration in the MF and the BF. The depressed effects on both forests occurred mostly in the warm-wet seasons and were correlated with a decrease in soil microbial activity and in fine root biomass caused by soil acidification under SAR. The sensitivity of the response of soil respiration to SAR showed an increasing trend with the progressive maturity of the three forests, which may result from their differences in acid buffering ability in soil and in litter layer. These results indicated that the depressed effect of acid rain on soil respiration in southern China may be more pronounced in the future in light of the projected change in forest maturity. However, due to the nature of this field study with chronosequence design and the related pseudoreplication for forest types, this inference should be read with caution. Further studies are needed to draw rigorous conclusions regarding the response differences among forests of different maturity using replicated forest types.
doi:10.1371/journal.pone.0062207
PMCID: PMC3633851  PMID: 23626790
6.  The UDP-Glucuronate Decarboxylase Gene Family in Populus: Structure, Expression, and Association Genetics 
PLoS ONE  2013;8(4):e60880.
In woody crop plants, the oligosaccharide components of the cell wall are essential for important traits such as bioenergy content, growth, and structural wood properties. UDP-glucuronate decarboxylase (UXS) is a key enzyme in the synthesis of UDP-xylose for the formation of xylans during cell wall biosynthesis. Here, we isolated a multigene family of seven members (PtUXS1-7) encoding UXS from Populus tomentosa, the first investigation of UXSs in a tree species. Analysis of gene structure and phylogeny showed that the PtUXS family could be divided into three groups (PtUXS1/4, PtUXS2/5, and PtUXS3/6/7), consistent with the tissue-specific expression patterns of each PtUXS. We further evaluated the functional consequences of nucleotide polymorphisms in PtUXS1. In total, 243 single-nucleotide polymorphisms (SNPs) were identified, with a high frequency of SNPs (1/18 bp) and nucleotide diversity (πT = 0.01033, θw = 0.01280). Linkage disequilibrium (LD) analysis showed that LD did not extend over the entire gene (r2<0.1, P<0.001, within 700 bp). SNP- and haplotype-based association analysis showed that nine SNPs (Q <0.10) and 12 haplotypes (P<0.05) were significantly associated with growth and wood property traits in the association population (426 individuals), with 2.70% to 12.37% of the phenotypic variation explained. Four significant single-marker associations (Q <0.10) were validated in a linkage mapping population of 1200 individuals. Also, RNA transcript accumulation varies among genotypic classes of SNP10 was further confirmed in the association population. This is the first comprehensive study of the UXS gene family in woody plants, and lays the foundation for genetic improvements of wood properties and growth in trees using genetic engineering or marker-assisted breeding.
doi:10.1371/journal.pone.0060880
PMCID: PMC3629030  PMID: 23613749
7.  Allelic Variation in PtGA20Ox Associates with Growth and Wood Properties in Populus spp 
PLoS ONE  2012;7(12):e53116.
Populus tomentosa is an economically important tree crop that produces wood for lumber, pulp, paper, and biofuels. Wood quality traits are likely to be strongly affected by the plant hormone gibberellic acid (GA), which regulates growth. GA20Ox encodes one of the major regulatory enzymes of GA biosynthesis and may therefore play a large role in growth and wood quality. Here, linkage disequilibrium (LD) studies were used to identify significant associations between single nucleotide polymorphisms (SNPs) within PtGA20Ox and growth and wood-quality traits of P. tomentosa. We isolated a full-length GA20Ox cDNA from Populus tomentosa by reverse transcription (RT)-PCR; this 1401 bp cDNA clone had an open reading frame of 1158 bp and encoded a protein of 385 amino acids. PtGA20Ox transcripts were maximally expressed in the mature xylem of vascular tissues, suggesting that PtGA20Ox is highly expressed and specifically associated with secondary xylem formation. Resequencing the PtGA20Ox locus of 36 individuals identified 55 SNPs, and the frequency of SNPs was 1/31 bp. The 29 most common SNPs (frequency>0.1) were genotyped in an association population (426 individuals) that was also phenotyped for key growth and wood quality traits. LD did not extend over the entire gene (r2<0.1, within 500 bp), demonstrating that a candidate-gene-based LD approach may the best way to understand the molecular basis underlying quantitative variation in this species. SNP- and haplotype-based association analyses indicated that four SNPs (false discovery rate Q<0.05) and 14 haplotypes (P<0.05) were significantly associated with growth and wood properties. The phenotypic variance explained by each SNP ranged from 3.44% to 14.47%. The SNP markers identified in this study can be applied to breeding programs for the improvement of growth and wood-property traits by marker-assisted selection.
doi:10.1371/journal.pone.0053116
PMCID: PMC3534044  PMID: 23300875
8.  Development and Application of Microsatellites in Candidate Genes Related to Wood Properties in the Chinese White Poplar (Populus tomentosa Carr.) 
Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are often preferred over random genomic markers because they represent variation in gene coding and/or regulatory regions. We characterized 544 genic SSR loci derived from 138 candidate genes involved in wood formation, distributed throughout the genome of Populus tomentosa, a key ecological and cultivated wood production species. Of these SSRs, three-quarters were located in the promoter or intron regions, and dinucleotide (59.7%) and trinucleotide repeat motifs (26.5%) predominated. By screening 15 wild P. tomentosa ecotypes, we identified 188 polymorphic genic SSRs with 861 alleles, 2–7 alleles for each marker. Transferability analysis of 30 random genic SSRs, testing whether these SSRs work in 26 genotypes of five genus Populus sections (outgroup, Salix matsudana), showed that 72% of the SSRs could be amplified in Turanga and 100% could be amplified in Leuce. Based on genotyping of these 26 genotypes, a neighbour-joining analysis showed the expected six phylogenetic groupings. In silico analysis of SSR variation in 220 sequences that are homologous between P. tomentosa and Populus trichocarpa suggested that genic SSR variations between relatives were predominantly affected by repeat motif variations or flanking sequence mutations. Inheritance tests and single-marker associations demonstrated the power of genic SSRs in family-based linkage mapping and candidate gene-based association studies, as well as marker-assisted selection and comparative genomic studies of P. tomentosa and related species.
doi:10.1093/dnares/dss031
PMCID: PMC3576656  PMID: 23213110
candidate gene-derived SSRs; cross-species transferability; in silico analysis of SSR variations; Populus tomentosa; single marker–trait association mapping
9.  Correction: Effects of Precipitation Increase on Soil Respiration: A Three-Year Field Experiment in Subtropical Forests in China 
PLoS ONE  2012;7(10):10.1371/annotation/1f49fc5e-e3f9-4b90-b555-97a54990ac3f.
doi:10.1371/annotation/1f49fc5e-e3f9-4b90-b555-97a54990ac3f
PMCID: PMC3935814
10.  MicroRNA-212 displays tumor-promoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1 
Molecular Biology of the Cell  2012;23(8):1423-1434.
Overexpression of microRNA-212 promoted cell cycle progression and cell proliferation, migration, and invasion in non–small cell lung cancer cells. PTCH1, a receptor of hedgehog pathway, is a functional target of miR-212. The role of miR-212 in cell proliferation may be mediated by PTCH1.
Dysexpression of microRNAs has been found in many tumors, including lung cancer. The hedgehog (Hh) signaling pathway plays an important role during normal development, and the abnormal regulation of its members has also been related to many tumors. However, little is known about the relationship between microRNA and the Hh pathway. In this paper, we report microRNA-212 (miR-212) playing a role in non-small cell lung cancer (NSCLC) and targeting PTCH1, a receptor of the Hh pathway. We found that miR-212 was up-regulated when cells were treated with 4ß-12-O-tetradecanoylphorbol-13-acetate (TPA). We ectopically expressed miR-212 in NSCLC cell lines to examine the influence of miR-212 overexpression. The results showed that overexpression of miR-212 in NSCLC cells promoted cell cycle progression and cell proliferation, migration, and invasion. The promoting effects of miR-212 on cell proliferation, migration, and invasion were partially reversed by the miR-212 inhibitor anti-miR-212. These results suggested that miR-212 might have tumor-promoting properties. Potential targets of miR-212 were predicted, and we showed tumor suppressor PTCH1 was a functional target of miR-212. PTCH1 may be responsible for the effect of miR-212 on cell proliferation. Altogether, our results indicated that miR-212 was involved in tumorigenesis, and the oncogenic activity of miR-212 in NSCLC cells was due, in part, to suppression of PTCH1.
doi:10.1091/mbc.E11-09-0777
PMCID: PMC3327317  PMID: 22357618
11.  Transcriptional profiling by cDNA-AFLP analysis showed differential transcript abundance in response to water stress in Populus hopeiensis 
BMC Genomics  2012;13:286.
Background
Drought is one of the main environmental factors limiting tree growth and productivity of plantation forests worldwide. Populus hopeiensis Hu et Chow is one of the most important commercial plantation tree species in China. However, the genes controlling drought tolerance in this species have not been identified or characterized. Here, we conducted differential expression analyses and identified a number of genes that were up- or downregulated in P. hopeiensis during water stress. To the best of our knowledge, this is the first comprehensive study of differentially expressed genes in water-stressed P. hopeiensis.
Results
Using the cDNA-AFLP detection technique, we used 256 primer combinations to identify differentially expressed genes in P. hopeiensis during water stress. In total, 415 transcript derived-fragments (TDFs) were obtained from 10× deep sequencing of 473 selected TDFs. Of the 415 TDFs, 412 were annotated by BLAST searches against various databases. The majority of these genes encoded products involved in ion transport and compartmentalization, cell division, metabolism, and protein synthesis. The TDFs were clustered into 12 groups on the basis of their expression patterns. Of the 415 reliable TDFs, the sequences of 35 were homologous to genes that play roles in short or long-term resistance to drought stress. Some genes were further selected for validation of cDNA-AFLP expression patterns using real-time PCR analyses. The results confirmed the expression patterns that were detected using the cDNA-AFLP technique.
Conclusion
The cDNA-AFLP technique is an effective and powerful tool for identifying candidate genes that are differentially expressed under water stress. We demonstrated that 415 TDFs were differentially expressed in water-stressed poplar. The products of these genes are involved in various biological processes in the drought response of poplar. The results of this study will aid in the identification of candidate genes of future experiments aimed at understanding this response of poplar.
doi:10.1186/1471-2164-13-286
PMCID: PMC3443059  PMID: 22747754
12.  Varied Pathways of Stage IA Lung Adenocarcinomas Discovered by Integrated Gene Expression Analysis 
Background: Discovery of the progression-associated genes and pathways in lung adenocarcinoma (LAD) has important implications in understanding the molecular mechanism of tumor development. However, few studies had been performed to focus on the changes of pathways in lung adenocarcinoma development using microarray expression profile.
Result: We performed a meta-analysis of 4 LAD microarray datasets encompassing 353 patients to reveal differentially expressed genes (DEGs) between normal lung tissues and LAD of different stages. Overall, 1 838 genes were found to be dys-regulated, and the adipogenesis, circadian rhythm, and Id pathways were significantly changed. Interestingly, most of the genes from the same gene family (such as Interleukin receptor, Matrix metallopeptidase, Histone cluster and Minichromosome maintenance complex component families) were found to be up-regulated (or down-regulated). Real-time PCR (qRT-PCR) was applied to validate the expression of randomly selected 18 DEGs in LAD cell lines. In the pathway analysis among stages, Oxidative stress, Glycolysis/Gluconeogenesis and Integrin-mediated cell adhesion pathways, which were involved in cancer cell proliferation and metastasis, were showed to be significantly regulated in stages other than IA.
Conclusion: Genes involved in adipogenesis and Id pathways might play important roles in development of LADs. The similar trend of expression of the gene family members suggested coordinate regulation in tumor progression. Three pathways (Oxidative stress, Glycolysis/Gluconeogenesis and Integrin-mediated cell adhesion pathways) significantly regulated in stages other than stage IA suggested that genes and pathways conferring invasive character might be activated in the preinvasive stage IB, while the Oxidative stress and the Glycolysis/Gluconeogenesis pathways might have strong connections to cisplatin-based chemotherapy. The insignificantly regulated three pathways in stage IA might be used in early-stage detection of LAD.
PMCID: PMC3088877  PMID: 21552421
meta-analysis; lung adenocarcinoma; pathway; sample size
13.  Potentials of Mean Force for Acetylcholine Unbinding from the Alpha7 Nicotinic Acetylcholine Receptor Ligand Binding Domain 
The nicotinic acetylcholine receptor is a prototype ligand-gated ion channel that mediates signal transduction in the neuromuscular junctions and other cholinergic synapses. The molecular basis for the energetics of ligand binding and unbinding is critical to our understanding of the pharmacology of this class of receptors. Here we used steered molecular dynamics to investigate the unbinding of acetylcholine from the ligand-binding domain of human alpha7 nicotinic acetylcholine receptor along four different predetermined pathways. Pulling forces were found to correlate well with interactions between acetylcholine and residues in the binding site during the unbinding process. From multiple trajectories along these unbinding pathways, we calculated the potentials of mean force for acetylcholine unbinding. Four available methods based on Jarzynski's equality were used and compared for their efficiencies. The most probable pathway was identified to be along a direction approximately parallel to the membrane. The derived binding energy for acetylcholine was in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human alpha7 nicotinic acetylcholine receptor. In addition, it is likely that several intermediate states exist along the unbinding pathways.
doi:10.1021/ja057292u
PMCID: PMC2546508  PMID: 16506783
14.  Electrostatic Properties of Cowpea Chlorotic Mottle Virus and Cucumber Mosaic Virus Capsids 
Biopolymers  2006;82(2):106-120.
Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca2+ can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca2+ ions can induce the capsid contraction and stabilize the native form.
doi:10.1002/bip.20409
PMCID: PMC2440512  PMID: 16278831
Computational Modeling; Supramolecular Assembly; Poisson-Boltzmann Equation; Electrostatic Potential; Simulation; Normal Mode Analysis; Swelling; Electrostatic Binding Energy
15.  Electrostatic Interaction between RNA and Protein Capsid in CCMV Simulated by a Coarse-grain RNA model and a Monte Carlo Approach 
Biopolymers  2004;75(4):325-337.
Although many viruses have been crystallized and the protein capsid structures have been determined by X-ray crystallography, the nucleic acids often can not be resolved. This is especially true for RNA viruses. The lack of information about the conformation of DNA/RNA greatly hinders our understanding of the assembly mechanism of various viruses. Here we combine a coarse-grain model and a Monte Carlo method to simulate the distribution of viral RNA inside the capsid of Cowpea Chlorotic Mottle Virus (CCMV). Our results show that there is very strong interaction between the N-terminal residues of the capsid proteins, which are highly positive-charged, and the viral RNA. Without these residues, the binding energy disfavors the binding of RNA by the capsid. The RNA forms a shell close to the capsid with the highest densities associated with the capsid dimers. These high-density regions are connected to each other in the shape of a continuous net of triangles. The overall icosahedral shape of the net overlaps with the capsid subunit icosahedral organization. Medium density of RNA is found under the pentamers of the capsid. These findings are consistent with experimental observations.
doi:10.1002/bip.20120
PMCID: PMC2426774  PMID: 15386271
CCMV; RNA coarse-grain model; Monte Carlo; Poisson Boltzmann Equation; electrostatic potential; simulation
16.  The Association of Tetrameric Acetylcholinesterase with ColQ Tail: A Block Normal Mode Analysis 
PLoS Computational Biology  2005;1(6):e62.
Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine in the neuromuscular junctions and other cholinergic synapses to terminate the neuronal signal. In physiological conditions, AChE exists as tetramers associated with the proline-rich attachment domain (PRAD) of either collagen-like Q subunit (ColQ) or proline-rich membrane-anchoring protein. Crystallographic studies have revealed that different tetramer forms may be present, and it is not clear whether one or both are relevant under physiological conditions. Recently, the crystal structure of the tryptophan amphiphilic tetramerization (WAT) domain of AChE associated with PRAD ([WAT]4PRAD), which mimics the interface between ColQ and AChE tetramer, became available. In this study we built a complete tetrameric mouse [AChET]4–ColQ atomic structure model, based on the crystal structure of the [WAT]4PRAD complex. The structure was optimized using energy minimization. Block normal mode analysis was done to investigate the low-frequency motions of the complex and to correlate the structure model with the two known crystal structures of AChE tetramer. Significant low-frequency motions among the catalytic domains of the four AChE subunits were observed, while the [WAT]4PRAD part held the complex together. Normal mode involvement analysis revealed that the two lowest frequency modes were primarily involved in the conformational changes leading to the two crystal structures. The first 30 normal modes can account for more than 75% of the conformational changes in both cases. The evidence further supports the idea of a flexible tetramer model for AChE. This model can be used to study the implications of the association of AChE with ColQ.
Synopsis
Acetylcholinesterase (AChE) breaks down acetylcholine in the neuromuscular junction and other cholinergic synapses to terminate neuronal signals. AChE exists as tetramers anchored by structural subunits to the cell membranes in the brain or the basal lamina in the neuromuscular junction. Based on a crystal structure of the tetramerization domain of AChE with a proline-rich attachment domain of the anchoring proteins, a symmetric model of the complex of AChE tetramer with the anchoring protein tail was constructed. Block normal mode analysis revealed the presence of several low-frequency, low-barrier normal modes corresponding to inter-subunit motions. Previous crystal structures of AChE tetramer could be rationalized using these normal modes. These low-frequency modes are due to the presence of a flexible hinge in the structure of AChE. This study paints a picture of a flexible AChE tetramer with different conformational states interconverting easily under physiological conditions, which has important implications on the function of AChE. In particular, AChE is not trapped in the compact tetramer structure, for which access of substrate to two of the active sites is somewhat limited. Rather, the tetramer fluctuates to expose all four of its active sites to ensure rapid removal of acetylcholine.
doi:10.1371/journal.pcbi.0010062
PMCID: PMC1285061  PMID: 16299589
17.  The Association of Tetrameric Acetylcholinesterase with ColQ Tail: A Block Normal Mode Analysis 
PLoS Computational Biology  2005;1(6):e62.
Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine in the neuromuscular junctions and other cholinergic synapses to terminate the neuronal signal. In physiological conditions, AChE exists as tetramers associated with the proline-rich attachment domain (PRAD) of either collagen-like Q subunit (ColQ) or proline-rich membrane-anchoring protein. Crystallographic studies have revealed that different tetramer forms may be present, and it is not clear whether one or both are relevant under physiological conditions. Recently, the crystal structure of the tryptophan amphiphilic tetramerization (WAT) domain of AChE associated with PRAD ([WAT]4PRAD), which mimics the interface between ColQ and AChE tetramer, became available. In this study we built a complete tetrameric mouse [AChET]4–ColQ atomic structure model, based on the crystal structure of the [WAT]4PRAD complex. The structure was optimized using energy minimization. Block normal mode analysis was done to investigate the low-frequency motions of the complex and to correlate the structure model with the two known crystal structures of AChE tetramer. Significant low-frequency motions among the catalytic domains of the four AChE subunits were observed, while the [WAT]4PRAD part held the complex together. Normal mode involvement analysis revealed that the two lowest frequency modes were primarily involved in the conformational changes leading to the two crystal structures. The first 30 normal modes can account for more than 75% of the conformational changes in both cases. The evidence further supports the idea of a flexible tetramer model for AChE. This model can be used to study the implications of the association of AChE with ColQ.
Synopsis
Acetylcholinesterase (AChE) breaks down acetylcholine in the neuromuscular junction and other cholinergic synapses to terminate neuronal signals. AChE exists as tetramers anchored by structural subunits to the cell membranes in the brain or the basal lamina in the neuromuscular junction. Based on a crystal structure of the tetramerization domain of AChE with a proline-rich attachment domain of the anchoring proteins, a symmetric model of the complex of AChE tetramer with the anchoring protein tail was constructed. Block normal mode analysis revealed the presence of several low-frequency, low-barrier normal modes corresponding to inter-subunit motions. Previous crystal structures of AChE tetramer could be rationalized using these normal modes. These low-frequency modes are due to the presence of a flexible hinge in the structure of AChE. This study paints a picture of a flexible AChE tetramer with different conformational states interconverting easily under physiological conditions, which has important implications on the function of AChE. In particular, AChE is not trapped in the compact tetramer structure, for which access of substrate to two of the active sites is somewhat limited. Rather, the tetramer fluctuates to expose all four of its active sites to ensure rapid removal of acetylcholine.
doi:10.1371/journal.pcbi.0010062
PMCID: PMC1285061  PMID: 16299589

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