Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A

Biological responses to histone methylation critically depend on the faithful readout and transduction of the methyl-lysine signal by "effector" proteins, yet our understanding of methyl-lysine recognition has so far been limited to the study of histone binding by chromodomain and WD40-repeat proteins. The double tudor domain of JMJD2A, a Jmjc domain-containing histone demethylase, binds methylated histone H3-K4 and H4-K20. We found that the double tudor domain has an interdigitated structure, and the unusual fold is required for its ability to bind methylated histone tails. The cocrystal structure of the JMJD2A double tudor domain with a trimethylated H3-K4 peptide reveals that the trimethyl-K4 is bound in a cage of three aromatic residues, two of which are from the tudor-2 motif, whereas the binding specificity is determined by side-chain interactions involving amino acids from the tudor-1 motif. Our study provides mechanistic insights into recognition of methylated histone tails by tudor domains and reveals the structural intricacy of methyl-lysine recognition by two closely spaced effector domains.     

Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle

CLC proteins transport chloride (Cl(-)) ions across cell membranes to control the electrical potential of muscle cells, transfer electrolytes across epithelia, and control the pH and electrolyte composition of intracellular organelles. Some members of this protein family are Cl(-) ion channels, whereas others are secondary active transporters that exchange Cl(-) ions and protons (H(+)) with a 2:1 stoichiometry. We have determined the structure of a eukaryotic CLC transporter at 3.5 angstrom resolution. Cytoplasmic cystathionine beta-synthase (CBS) domains are strategically positioned to regulate the ion-transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate residue changes conformation and suggests a basis for 2:1 Cl(-)/H(+) exchange and a simple mechanistic connection between CLC channels and transporters.     

Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 Å resolution

Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH (bromo-associated homology) domain and the nucleosome core particle at 3.0 angstrom resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.     

Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP

Polyadenylate [poly(A)] polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs). The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP). Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site. The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides. The first two domains are functionally analogous to polymerase palm and fingers domains. The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer. In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1. One occupies the position of the incoming base, prior to its addition to the mRNA chain. The other is believed to occupy the position of the 3' end of the mRNA primer.     

The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition

When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 angstroms and compare it with a structure of the motor domain alone at 2.7 angstroms. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled coil. This "double lockdown," by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release adenosine diphosphate. This autoinhibition mechanism could extend to some other kinesins.     

烟草H2A的序列分析、原核表达载体构建及诱导表达

[目的]对H2A进行序列分析及蛋白结构域分析,并将其克隆入p GEX4T-1原核表达载体进行诱导表达。[方法]以p GADT7-Rec-H2A质粒为模板,通过PCR扩增烟草H2A(3~140 Aa),将其插入原核表达载体p GEX-4T-1中,并将重组载体转入BL21(DE3)中,诱导其表达。[结果]构建了烟草H2A基因全长和p GEX4T-1载体大片段连接的融合表达载体,并成功地诱导其表达。[结论]该研究为运用Pull Down确定Nt Tkr与候选蛋白之间的体外互作进而确定Nt Tkr与蛋白互作的关键结构域奠定了试验基础。     

Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function

Mutation of the VHL tumor suppressor is associated with the inherited von Hippel-Lindau (VHL) cancer syndrome and the majority of kidney cancers. VHL binds the ElonginC-ElonginB complex and regulates levels of hypoxia-inducible proteins. The structure of the ternary complex at 2.7 angstrom resolution shows two interfaces, one between VHL and ElonginC and another between ElonginC and ElonginB. Tumorigenic mutations frequently occur in a 35-residue domain of VHL responsible for ElonginC binding. A mutational patch on a separate domain of VHL indicates a second macromolecular binding site. The structure extends the similarities to the SCF (Skp1-Cul1-F-box protein) complex that targets proteins for degradation, supporting the hypothesis that VHL may function in an analogous pathway.     

Mechanism of two classes of cancer mutations in the phosphoinositide 3-kinase catalytic subunit

Many human cancers involve up-regulation of the phosphoinositide 3-kinase PI3Kalpha, with oncogenic mutations identified in both the p110alpha catalytic and the p85alpha regulatory subunits. We used crystallographic and biochemical approaches to gain insight into activating mutations in two noncatalytic p110alpha domains-the adaptor-binding and the helical domains. A structure of the adaptor-binding domain of p110alpha in a complex with the p85alpha inter-Src homology 2 (inter-SH2) domain shows that oncogenic mutations in the adaptor-binding domain are not at the inter-SH2 interface but in a polar surface patch that is a plausible docking site for other domains in the holo p110/p85 complex. We also examined helical domain mutations and found that the Glu545 to Lys545 (E545K) oncogenic mutant disrupts an inhibitory charge-charge interaction with the p85 N-terminal SH2 domain. These studies extend our understanding of the architecture of PI3Ks and provide insight into how two classes of mutations that cause a gain in function can lead to cancer.     

Electron spin resonance in studies of membranes and proteins

We provide a review of current electron spin resonance (ESR) techniques for studying basic molecular mechanisms in membranes and proteins by using nitroxide spin labels. In particular, nitroxide spin label studies with high-field/high-frequency ESR and two-dimensional Fourier transform ESR enable one to accurately determine distances in biomolecules, unravel the details of the complex dynamics in proteins, characterize the dynamic structure of membrane domains, and discriminate between bulk lipids and boundary lipids that coat transmembrane peptides or proteins; these studies can also provide time resolution to studies of functional dynamics of proteins. We illustrate these capabilities with recent examples.     

A common fold mediates vertebrate defense and bacterial attack

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.     

The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses

The transition from the expression of alpha, the first set of five herpes simplex virus genes expressed after infection, to beta and gamma genes, expressed later in infection, requires the participation of infected cell protein 4 (alpha 4), the major viral regulatory protein. The alpha 4 protein is present in complexes formed by proteins extracted from infected cells and viral DNA fragments derived from promoter domains. This report shows that the alpha 4 protein forms specific complexes with DNA fragments derived from 5' transcribed noncoding domains of late (gamma 2) genes whose expression requires viral DNA synthesis as well as functional alpha 4 protein. Some of the DNA fragments to which alpha 4 binds do not contain homologs of the previously reported DNA binding site consensus sequence, suggesting that alpha 4 may recognize and interact with more than one type of DNA binding site. The alpha 4 proteins can bind to DNA directly. A posttranslationally modified form of the alpha 4 protein designated alpha 4c differs from the alpha 4a and alpha 4b forms with respect to its affinity for DNA fragments differing in the nucleotide sequences of the binding sites.     

大麦TIFY基因家族成员鉴定及表达分析

【目的】对大麦TIFY基因家族成员进行鉴定及表达分析,为进一步探究TIFY基因家族在大麦生长发育与胁迫响应中的作用机理打下基础。【方法】基于TIFY家族蛋白的保守域特征,利用HMMER从大麦中鉴定TIFY基因家族成员,利用采用生物信息学软件对其理化性质、保守基序、特征结构域、顺式作用元件、基因结构、系统进化及表达模式进行预测分析。【结果】从大麦中鉴定出15个HvTIFYs基因（HvTIFY1～HvTIFY15）,分布于5条染色体上,且大多数基因在染色体上成簇分布。15个HvTIFYs蛋白均具有TIFY家族蛋白的特征结构域（TIFY）,根据所含保守结构域的不同,可分为ZML（4个）和JAZ亚族（11个）,且亲水性蛋白（14个）和偏碱性蛋白（11个）居多,但均定位于细胞核;二级结构相似度较高,均由α-螺旋、β-转角和无规则卷曲组成,除HvTIFY7蛋白外,其余蛋白二级结构所占比排序:无规则卷曲>α-螺旋>β-转角。HvTIFYs基因结构存在明显差异,其中,JAZ亚族11个基因的内含子数为0~6; ZML亚族4个基因的内含子数为6～7个,系统发育进化树上相邻分支的基因具有较相似的基因结构。HvTIFYs基因启动子区域富含光、激素和胁迫等顺式作用元件,种类及分布均呈多样性。5个物种的79条TIFY蛋白分为4个组,恰好与TIFY家族的4个亚族对应,其中,ZML、TIFY和JAZ亚族包含单、双子叶植物的TIFY蛋白,而PPD亚族仅含有双子叶植物的TIFY蛋白。15个HvTIFYs基因在不同组织器官中的表达量存在明显差异,其中HvTIFY1、HvTIFY2和HvTIFY8基因在8个组织中的表达量均较高,HvTIFY10和HvTIFY15基因表达量中等,HvTIFY6基因表达量较低; HvTIFY11基因不表达。15个基因在根的不同组织中对盐胁迫的敏感程度不同。【结论】从大麦中鉴定出的15个HvTIFYs基因存在一定的功能分化,具有明显的组织和时空特异性,推测其在大麦逆境响应和激素调节中具有重要调控作用。     

陆地棉两个GhBI-1基因的生物信息学分析

BI-1基因是广泛存在于真核生物、原核生物和病毒中的一个保守性基因,具有抑制细胞凋亡的作用。本研究对克隆到的两个陆地棉BI-1基因——GhBI-1a和GhBI-1b进行生物信息学分析,发现其与拟南芥BI-1有很高的同源性,尤其羧基尾极为相似。GhBI-1a有6个跨膜区域,GhBI-1b有7个跨膜区域,它们的二级结构也与其他物种的BI-1相似,跨膜区域基本都是α螺旋。     

大白菜GIF蛋白家族的生物信息学分析

GIF(GRF-interacting factor)家族是一类含有SNH和QG结构域的蛋白质,可与GRF(Growth reg-ulating factor)转录因子蛋白相结合形成功能复合体,通过促进和维持细胞的分裂能力参与调控植物叶器官的发育。本研究系统鉴定了5个大白菜的GIF基因,并对这些基因编码的蛋白质序列进行了保守性和系统进化分析,最后对BrGIF1基因的表达进行了分析。结果表明,所有的大白菜和拟南芥GIF蛋白家族成员都具有高度保守的SNH和QG结构域。在进化上,GIF蛋白家族可分为两个不同的亚家族,并且这种特征在大白菜和拟南芥分离之前就已经形成。在表达模式上,BrGIF1基因在具有较大叶球的白菜自交系以及具有较强细胞分裂能力的组织中的转录表达水平较高。另外,BrGIF1基因的表达受到NAA的诱导和ABA的抑制。这些结果表明大白菜GIF蛋白可能具有和拟南芥GIF蛋白相似的生物学功能,在调控植物器官发育中具有重要作用。     

Arbuscular mycorrhizal associations and the major regulators

Plants growing in natural soils encounter diverse biotic and abiotic stresses and have adapted with sophisticated strategies to deal with complex environments such as changing root system structure, evoking biochemical responses and recruiting microbial partners. Under selection pressure, plants and their associated microorganisms assemble into a functional entity known as a holobiont. The commonest cooperative interaction is between plant roots and arbuscular mycorrhizal (AM) fungi. About 80% of terrestrial plants can form AM symbiosis with the ancient phylum Glomeromycota. A very large network of extraradical and intraradical mycelium of AM fungi connects the underground biota and the nearby carbon and nutrient fluxes. Here, we discuss recent progress on the regulators of AM associations with plants, AM fungi and their surrounding environments, and explore further mechanistic insights.     

家蚕DNA甲基转移酶功能预测及其在BmNPV感染下的表达特征

【目的】明确家蚕DNA甲基转移酶基因(BmDNMT)生物信息学基本特征及其在家蚕核型多角体病毒(BmNPV)感染前后的表达情况,揭示DNA甲基化在家蚕抗病毒免疫方面的作用机制,为挖掘家蚕抗病毒标志基因和药物靶标蛋白等提供理论依据。【方法】通过OrthoDB、MultiLoc2、SherLoc2、PSORTII、SMART、SWISS-MODEL及Schr?dinger等在线生物信息学分析软件进行BmDNMT蛋白氨基酸同源比对、系统进化分析、亚细胞定位、功能结构域预测、三级结构同源建模及小分子配体对接口袋预测,并采用实时荧光定量PCR分析BmDNMT基因在家蚕感染BmNPV后不同时间不同组织中的时空表达特征。【结果】BmDNMT基因包含BmDNMT1和BmDNMT2,分别位于家蚕8号染色体和11号染色体上,其推导BmDNMT氨基酸序列均与烟草天蛾DNMT氨基酸序列的亲缘关系最近。BmDNMT1蛋白大量存在于细胞核,少量分布在细胞质;而BmDNMT2蛋白大量存在于细胞质,少量分布在细胞核及线粒体。BmDNMT1蛋白的功能结构域多于BmDNMT2蛋白,二者均含有DNA甲基化酶功能结构域,且具有潜在药物结合口袋。BmNPV感染会影响BmDNMT1和BmDNMT2基因在家蚕不同组织中表达差异,BmNPV感染4 h后这2个基因在家蚕中肠的表达差异已非常明显,说明家蚕DNA甲基化可能在病毒感染早期已发生,且中肠可能是最早响应的组织。BmNPV感染后,BmDNMT1和BmDNMT2基因的相对表达量和表达趋势并不一致。【结论】BmDNMT1和BmDNMT2基因的染色体位置、亚细胞定位及功能结构域等存在差异,BmNPV感染后二者在家蚕不同组组织中的相对表达量和表达趋势也不一致,推测BmDNMT1和BmDNMT2基因在家蚕DNA甲基化过程中行使不同的功能。BmDNMT1和BmDNMT2蛋白三维结构具有潜在的药物结合口袋,可能是直接影响DNA甲基化状态的药物靶标。