β肾上腺素受体的结构与功能域

β肾上腺素受体具有视紫红质样结构,包括由膜两侧亲水环相互联结7个疏水性跨膜α螺旋结构,N端无信号序列而含有2个N-糖基化位点,C端富含丝氨酸和苏氨酸残基.7个跨膜结构构成配基结合位点.β受体细胞膜内侧环状序列形成两亲α螺旋结构,与G蛋白相互作用.C端及第3个内侧环的丝氨酸及苏氨酸残基构成受体磷酸化位点,参与受体功能调控.     

P2X4受体的结构功能关系研究进展

嘌呤能受体P2X4是三磷酸腺苷(ATP)-门控的阳离子通道,对生物体内多种重要生命活动起一定的调节作用。二次跨膜的三聚体通道P2X4受体的三维空间组成是由胞外结构域、跨膜域及胞内N-、C-端组成。ATP的三磷酸基团能被位于亚基界面的ATP结合口袋的带正电氨基酸特异性识别,嘌呤环则被疏水氨基酸和部分氨基酸的主链氧所识别。P2X4受体激活后,胞外阳离子更多是通过侧窗路径进入细胞内。就P2X4受体的空间结构、配体的识别、离子通透途径及门控机制作一综述。     

肝细胞生长因子Kringle的三维结构模建及与功能的关系

利用同源模建的方法模拟得到了肝细胞生长因子4个Kringle域的三维结构。结果表明,HGFKringle与纤溶酶原Kringle的氨基酸序列具有较高的同源性,其功能区附近的序列比较保守。HGF的Kringlel和3与其它具有Lys结合功能的Kringle相比,功能区的残基发生了变化,可能丧失了结合Lys的功能,而2和4仍具有一定的该功能。根据Kringle 1的模建结构,推测该Kringle功能区的结构为一个通道,该通道的底部和一侧有部分疏水残基,同时两侧还分布着少量酸性或碱性残基,该通道可能具有结合特定肽链的功能,从而与Kringle 2一起实现HGF与受体结合的作用。     

乳腺癌易感蛋白BRCA1的BRCT2结构域染色质伸展活性的定位

40 %～ 5 0 %的遗传性乳腺癌和至少 80 %的既有乳腺癌又有卵巢癌家族史的患者是由BRCA1突变引起的 .BRCA1C末端含有 2个BRCT结构域 (BRCT1和BRCT2 ) ,它们与BRCA1的重要功能密切相关 .许多乳腺癌易感突变发生在BRCA1的BRCT结构域中 .利用染色质结构检测技术表明 ,BRCT结构域具有染色质伸展活性 .利用缺失突变技术构建了 6种BRCT2结构域 (175 6～ 185 2位氨基酸残基 )缺失突变体并将BRCT2结构域中与染色质伸展相关的重要区域定位到 175 6～ 180 8之间的氨基酸残基 ;用丙氨酸扫描技术构建了 6种BRCT2结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到 1784～ 1788之间的VQLCG .BRCT2结构域的定位有助于预测BRCT2结构域突变后发生乳腺癌的风险 ,也为进一步研究BRCT2结构域的功能机制提供了有用的材料 .     

光肩星天牛气味结合蛋白AglaOBP12同源建模及与乙酸-顺-3-己烯酯的分子对接研究

【目的】研究光肩星天牛Anoplophora glabripennis气味结合蛋白AglaOBP12与寄主植物挥发物乙酸-顺-3-己烯酯的相互作用机制,为利用化学生态手段调控光肩星天牛行为提供理论依据。【方法】采用同源建模预测AglaOBP12三维结构,虚拟氨基酸突变构建两个突变子,利用Molegro Virtual Docker程序进行分子对接研究AglaOBP12与乙酸-顺-3-己烯酯的结合模式。模型的合理性评价采用GMQE、QMEAN、ramachandran图和Verify-3D。【结果】AglaOBP12的三维结构由6个α-螺旋构成且形成锥形疏水口袋结构。6个保守半胱氨酸在螺旋结构之间形成稳定结构的3个二硫键。AglaOBP12的C端疏水性氨基酸位于结合口袋的出口并对口袋形成一定的遮挡。乙酸-顺-3-己烯酯位于疏水口袋中与疏水性氨基酸发生作用,而亲水头部羰基氧原子则与Asn123产生氢键。在与突变子N123A的对接中,乙酸-顺-3-己烯酯更接近疏水口袋的出口,乙酸-顺-3-己烯酯与C端Phe135形成氢键。而在与突变子F135E/L136E/V137E的对接中,乙酸-顺-3-己烯酯位于疏水口袋较深处,但未发现与Asn123氢键作用,相比野生型,两个突变子的对接空间能和范德华尔能增大,结合稳定性下降。【结论】乙酸-顺-3-己烯酯位于AglaOBP12疏水口袋并通过氢键与Asn123形成稳定的复合物,AglaOBP12的Asn123和C端疏水氨基酸对结合乙酸-顺-3-己烯酯具有重要作用。     

乳腺癌易感蛋白BRCA1的BRCT1结构域染色质伸展活性的定位

乳腺癌易感基因BRCA1（Breast cancer susceptibility gene 1）在乳腺癌的发生、发展中起重要作用。BRCA1 C末端含有2个BRCT结构域（BRCT1和BRCT2）,许多乳腺癌易感突变发生在BRCA1的BRCT结构域中。利用染色质结构检测技术表明,BRCT结构域具有染色质伸展活性。本文利用缺失突变技术构建了6种BRCT1结构域（1642-1736 aa）缺失突变体并将BRCT1结构域中与染色质伸展相关的重要区域定位到1691-1721之间的氨基酸残基;用丙氨酸扫描技术构建了10种BRCT1结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到1707-1711之间的IAGGK。利用定位的重要区域进行Blast分析,结果找到一新型同源蛋白质。BRCT1结构域的定位有助于预测BRCT1结构域突变后发生乳腺癌的风险,也为进一步研究BRCT1结构域的功能机制提供了有用的材料。     

t-PA K1区的同源模建及Kringle区与赖氨酸相互作用的研究

利用同源模建方法预测了t-PAK1区的三维结构。通过结构叠合确定了t-PAK1、K2区,纤溶酶原K1、K4区及UKK区的赖氨酸结合口袋。静电势计算及疏水性分析表明,在t-PAK2区以及纤溶酶原K1、K4区与纤维蛋白裸露的赖氨酸之间存在明显的静电势互补和疏水面契合。确定了影响Kringle区结合口袋与赖氨酸亲和的重要氨基酸,分析了t-PAK1区、UKK区不能结合赖氨酸的原因,由此设计了具有赖氨酸亲和力的新型t-PAK1区及UKK区突变体。利用模拟残基突变技术预测了突变体的结构变化,分析了突变后t-PAK1区及UKK区与赖氨酸亲和力的变化,初步在理论上肯定了设计方案的合理性。     

半乳糖凝集素糖结合的新模式：一个糖结合域的双重配体结合

半乳糖凝集素家族通过糖识别结构域(CRD)可以专一性识别和结合含β-半乳糖的多糖配体来发挥其生物学功能.到目前发现的CRD对β-半乳糖的识别模式是非常保守的,在结构已知的半乳糖凝集素结构中,一个CRD只能结合一个多糖配体分子.最近,通过对人源半乳糖凝聚素-3 CRD与对硝基TF二糖(TFN)复合物的晶体结构解析首次发现,一个CRD可以同时结合2个TFN分子.与这2个TFN分子有双向结合的残基突变体E165A结构分析显示,一个残基的突变引起的结构上的微小变化会使结合位点2丧失结合糖底物的能力,而位点1的配体结合却不受影响.这表明,结合位点1对糖底物保守的识别和结合是基本的、主要的,而结合位点2对于糖有条件的结合,是额外的、次要的.序列比对和立体化学分析显示,参与新位点2结合的关键残基在其他半乳糖凝集素分子中都是保守的,而它们参与糖配体结合并不常见,表明它们作用的发挥是有条件的.可能在复杂寡聚结构的情况下,如有多重分支结构,双重结合位点将有利于对这类配体分子的辨识和结合,已有一系列研究报道,具有分支结构的寡糖与半乳糖分子的亲和势明显高于单价糖配体,与上述分析相一致.对这类双重位点糖结合的可能生物学意义进行了讨论.     

HIV-1跨膜蛋白gp41与N-取代吡咯衍生物的结合模式研究

采用分子对接,分子动力学(MD)模拟和分子力学/泊松-波尔兹曼溶剂可有面积方法与分子力学/广义伯恩溶剂可及面积方法(MM-PBSA/MM-GBSA),预测两种N-取代吡咯衍生物与HIV-1 跨膜蛋白gp41疏水口袋的结合模式与作用机理．分子对接采用多种受体构象,并从结果中选取几种可能的结合模式进行MD 模拟,然后通过MM-PBSA计算结合能的方法识别最优的结合模式. MM-PBSA计算结果表明,范德华相互作用是结合的主要驱动力,而极性相互作用决定了配体在结合过程中的取向．进一步的结合能分解显示,配体的羧基与gp41残基Arg579的静电相互作用对结合有重要贡献．上述工作为进一步优化N-取代吡咯衍生物类的HIV-1融合抑制剂建立了良好的理论基础．     

Hsp70核苷酸结合域突变体影响酶活的分子动力学模拟研究

分子伴侣蛋白Hsp70氮端核苷酸结合域（NBD, nucleotide-binding domain）的ATP酶活性变化对其行使分子伴侣功能具有重要作用。本文采用分子动力学模拟方法研究酵母分子伴侣Hsp70氮端NBD内残基A17,R23,G32和R167点突变对其ATP酶活性区域构象影响及功能关系。结果表明,突变体A17V,T23H,G32S的ATP结合口袋袋口的loopl（第一个转角,连接p1与p2）结构柔性增强,活性残基T11侧链明显向内移动,从而更加接近ATP的γ-磷酸基团,更容易使ATP水解。这可能蕞终导致ATP酶活性增强,从而引起分子伴侣功能的变化。     

Crystal structure of the BARD1 BRCT domains

The interaction of the breast tumor suppressor BRCA1 with the protein BARD1 results in the formation of a heterodimeric complex that has ubiquitin ligase activity and plays central roles in cell cycle checkpoint control and DNA repair. Both BRCA1 and BARD1 possess a pair of tandem BRCT domains that interact in a phosphorylation-dependent manner with target proteins. We determined the crystal structure of the human BARD1 BRCT repeats (residues 568-777) at 1.9 A resolution. The composition and structure of the BARD1 phosphoserine-binding pocket P1 are strikingly similar to those of the BRCA1 and MDC1 BRCT domains, suggesting a similar mode of interaction with the phosphate group of the ligand. By contrast, the BARD1 BRCT selectivity pocket P2 exhibits distinct structural features, including two prominent histidine residues, His685 and His686, which may be important for ligand binding. The protonation state of these histidines has a marked effect on the calculated electrostatic potential in the vicinity of P2, raising the possibility that ligand recognition may be regulated by changes in pH. Importantly, the BARD1 BRCT structure provides insights into the mechanisms by which the cancer-associated missense mutations C645R, V695L, and S761N may adversely affect the structure and function of BARD1.     

Crystal structure of the N-terminal region of human Topoisomerase IIβ binding protein 1

Human DNA Topoisomerase IIβ binding protein 1 (TopBP1) is a modulating protein that plays an essential role in the response to DNA damage. The N-terminal region of TopBP1, which contains predicted BRCA1-carboxy terminal (BRCT) domains 1 and 2, binds to Rad9, a component of the cell cycle checkpoint clamp Rad9-Hus1-Rad1 complex. Here, we report the crystal structure of the TopBP1 N-terminal region (residues 1-290) at 2.4 Å resolution. Interestingly, in addition to the predicted tandem BRCT1-2 repeats (residues 103-284), residues 7-98 form a previously unreported BRCT domain (here, BRCT0). In contrast to both BRCT1 and BRCT2, which possess the conventional phosphopeptide binding residues within a surface pocket, the corresponding pocket in BRCT0 is largely hydrophobic. Structural comparisons together with peptide binding studies indicate that the tandem BRCT1-2 domains are the binding region for phosphorylated Ser387 in Rad9.     

Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra α-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5′-phosphate recognition by the BRCT domains of bacterial NAD⁺-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions.     

NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains.

Mammalian telomeres are composed of long tandem arrays of double-stranded telomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRap1p, which has a central DNA-binding domain that contains two structurally related Myb domains connected by a long linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 contained only one recognizable Myb motif in the center of the protein. Furthermore, while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding ability. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determined the solution structure of the Myb domain of hRap1 by NMR. It contains three helices maintained by a hydrophobic core. The architecture of the hRap1 Myb domain is very close to that of each of the Myb domains from TRF1, scRap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lack of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact instead with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds of all Myb domains resemble each other closely, the function of each Myb domain depends on the amino acid residues that are located on the surface of each protein.     

Structural basis for phosphorylation-dependent signaling in the DNA-damage response.

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.     

Interactions between BRCT repeats and phosphoproteins: tangled up in two

The C-terminal region of the breast-cancer-associated protein BRCA1 contains a pair of tandem BRCA1 C-terminal (BRCT) repeats that are essential for the tumour suppressor function of the protein. Similar repeat sequences have been identified in many proteins that seem to mediate cellular mechanisms for dealing with DNA damage. The BRCT domain in BRCA1 has been recently shown to constitute a module for recognizing phosphorylated (phospho-) peptides, with a recognition groove that spans both BRCT repeats. The fact that many other BRCT-containing proteins have phospho-peptide binding activity suggests that BRCT repeats might mediate phosphorylation-dependent protein-protein interactions in processes that are central to cell-cycle checkpoint and DNA repair functions.     

A Pocket on the Surface of the N-Terminal BRCT Domain of Mcph1 Is Required to Prevent Abnormal Chromosome Condensation

Mcph1 is mutated in autosomal recessive primary microcephaly and premature chromosome condensation (PCC) syndrome. Increased chromosome condensation is a common feature of cells isolated from patients afflicted with either disease. Normal cells depleted of Mcph1 also exhibit PCC phenotype. Human Mcph1 contains three BRCA1-carboxyl terminal (BRCT) domains, the first of which (Mcph1N) is necessary for the prevention of PCC. The only known disease-associated missense mutation in Mcph1 resides in this domain (T27R). We have determined the X-ray crystal structure of human Mcph1N to 1.6 Å resolution. Compared with other BRCT domain structures, the most striking differences are an elongated, ordered β1-α1 loop and an adjacent hydrophobic pocket. This pocket is in the equivalent structural position to the phosphate binding site of BRCT domains that recognize phospho-proteins, although the phosphate-binding residues are absent in Mcph1N. Mutations in the pocket abrogate the ability of full-length Mcph1 to rescue the PCC phenotype of Mcph1^−/− mouse embryonic fibroblast cells, suggesting that it forms an essential part of a protein-protein interaction site necessary to prevent PCC.     

The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.