Functional divergence in the Arabidopsis LOB-domain gene family

The Arabidopsis LOB-domain (LBD) gene family is composed by 43 members divided in two classes based on amino acid conservation within the LOB-domain. The LOB domain is known to be responsible for DNA binding and protein-protein interactions. There is very little functional information available for most genes in the LBD family and many lbd single mutants do not exhibit conspicuous phenotypes. One plausible explanation for the limited loss-of-function phenotypes observed in this family is that LBD genes exhibit significant functional redundancy. Here we discuss an example of one phylogenetic subgroup of the LBD family, in which genes that are closely related based on phylogeny exhibit distinctly different expression patterns and do not have overlapping functions. We discuss the challenges of using phylogenetic analyses to predict redundancy in gene families.     

鉴别杰多霉素生物合成后修饰氧化酶JadH中参与底物结合或催化的关键残基

JadH是羟化脱水双功能酶,参与杰多霉素生物合成中的聚酮后修饰反应,将2,3-dehydro-UWM6催化为dehydrorabelomycin。为了分析杰多霉素生物合成途径中后修饰氧化酶JadH结合、催化底物的关键氨基酸,构建了JadH与底物复合物的三维结构模型。利用该模型并结合JadH同源蛋白氨基酸序列比对分析,推测出JadH活性中心中可能参与底物结合或催化的关键氨基酸(R50、G51、L52、G53、F100、R221、I223、P295和G298)。通过定点突变及体外酶学实验对这些位点的突变体的催化活性进行评价,结果显示这些突变株活性均显著低于野生型,表明这9个氨基酸是JadH参与底物结合或催化的关键氨基酸。     

拟南芥甲基结合蛋白基因AtMBP11启动子在种子形成和萌发中的功能鉴定

为研究拟南芥甲基结合蛋白基因AtMBP11在种子形成和萌发过程中的调控模式,克隆拟南芥AtMBP11启动子,将其替换植物表达载体pBI121的35S启动子序列,转入拟南芥基因组中.转基因拟南芥后代卡那霉素抗性发生分离,选取具有3∶1分离比的后代自交,产生纯合的具有单拷贝插入的后代.转基因后代GUS染色结果表明,新克隆的MBP启动子控制基因在种子、花药和花粉中高效表达.通过对AtMBP11核心启动子缺失分析表明,G-box元件是主要功能元件.     

NIRF泛素化p53的作用过程中NIRF与p53结合域的测定

NIRF(Np95/ICBP90-like RING finger protein)是2002年发现的一种核蛋白,其功能涉及细胞增殖调节、蛋白多聚泛素化降解、细胞癌变进程控制等领域．已有研究报道,NIRF能与p53相互作用, NIRF本身也是一个高度调节蛋白,在细胞正常的生理状态下发挥泛素化E3连接酶的作用,结合p53并将其降解,但NIRF与p53结合的蛋白结合域目前尚不清楚．本文研究证明,NIRF能与p53结合成复合体参与泛素化蛋白降解途径,并测定出NIRF与p53结合的区域．为了检测NIRF的蛋白结合域,将空载体和NIRF缺失突变体质粒分别转染于HEK293细胞,蛋白表达水平通过Western印迹用两种抗体分别检测. 结果显示,所有的突变体都能在细胞中表达,并且两种抗体检测结果完全一致. 同时,免疫共沉淀技术用于进一步分析实验结果. 由于泛素化蛋白通常伴随蛋白酶体通路介导的降解,免疫共沉淀的蛋白纯化过程中用蛋白酶体抑制剂MG-132以抑制蛋白降解. 本研究结果显示,NIRF 通过PHD区域与p53形成复合体. 该复合体可能参与蛋白分选、蛋白降解、DNA修复以及细胞凋亡等一系列重要的细胞活动,从而形成与细胞增殖相关的新的信号通路,在肿瘤的发生发展中可能发挥某种程度的作用．     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

Structural polymorphism of oligomeric adiponectin visualized by electron microscopy

Adiponectin, a macromolecular complex similar to the members of the C1q and other collagenous homologues, elicits diverse biological functions, including anti-diabetes, anti-atherosclerosis, anti-inflammation and anti-tumor activities, which have been directly linked to the high molecular weight (HMW) oligomeric structures formed by multiples of adiponectin trimers. Here, we report the 3-D reconstructions of isolated full-length, recombinant murine C39A adiponectin trimer and hexamer of wild-type trimers (the major HMW form) determined by single-particle analysis of electron micrographs. The pleiomorphic ensemble of collagen-like stretches of the trimers leads to a dynamic structure of HMW that partition into two major classes, the fan-shaped (class I) and bouquet-shaped (class II). In both of these, while the N termini cluster into a compact ellipsoid-shaped (∼ 60 Å × 45 Å × 45 Å) volume, the collagenous domains assume a variety of arrangements. The domains are splayed by up to ∼ 90° in class I, can form a close-packed, up to ∼ 100 × 40 Å cylindrical assembly in class II, which can house about half of the 66 putative collagen-like sequence and the rest, tethered to the trimeric globular domains at the C terminus, are highly dynamic. As a result, the globular domains elaborate a variety of arrangements, covering an area of up to ∼ 4.9 × 10⁵ Ų and up to ∼ 320 Å apart, some of which were captured in reconstructions of class II. Our reconstructions suggest that the N-terminal structured domain, agreeing approximately with the expected volume for the octadecameric assembly of the terminal 27 amino acids, is crucial to the formation of the functionally active HMW. On the other hand, conformational flexibility of the trimers at the C terminus can allow the HMW to access and cluster disparate target ligands binding to the globular domains, which may be necessary to activate cellular signaling leading to the remarkable functional diversity of adiponectin.     

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform ¹³C and ¹⁵N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.     

Incorporation of the Arc1p tRNA-binding domain to the catalytic core of MetRS can functionally replace the yeast Arc1p-MetRS complex

The catalytic core of methionyl-tRNA synthetase (MetRS) is conserved among all life kingdoms but, depending on species origin, is often linked to non-catalytic domains appended to its N- or C-terminus. These domains usually contribute to protein-protein or protein-tRNA interactions but their exact biological role and evolutionary purpose is poorly understood. Yeast MetRS contains an N-terminal appendix that mediates its interaction with the N-terminal part of Arc1p. Association with Arc1p controls the subcellular distribution of MetRS. Furthermore, the C-terminal part of Arc1p harbors a conserved tRNA-binding domain (TRBD) required for the Arc1p-dependent stimulation of the catalytic activity of MetRS. The same TRBD is found directly fused to catalytic domains of plant and nematode MetRS as well as human tyrosyl-tRNA synthetase. To investigate the purpose of Arc1p-MetRS complex formation in yeast, we tested the ability of TRBD to assist the function of MetRS independently of Arc1p. We attached the TRBD directly to the C-terminus of the MetRS catalytic core (MC) by constructing the chimeric protein MC-TRBD. The effect of MC-TRBD expression on yeast cell growth as well as its localization and in vitro aminoacylation activity were analyzed and compared to that of MC alone or wild-type MetRS, both in the absence or presence of Arc1p. We show that MC-TRBD exhibits improved enzymatic activity and can effectively substitute the MetRS-Arc1p binary complex in vivo. Moreover, MC-TRBD, being exclusively cytoplasmic, also mimics the MetRS-Arc1p complex in terms of subcellular localization. Our results suggest that the sole role of the N-terminal appended domain of yeast MetRS is to mediate the indirect association with the TRBD, which, nevertheless, can also function effectively in vivo when directly fused to the catalytic MetRS core.     

Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its ∼ 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.     

Structural insights into the recognition of peroxisomal targeting signal 1 by Trypanosoma brucei peroxin 5

Glycosomes are peroxisome-like organelles essential for trypanosomatid parasites. Glycosome biogenesis is mediated by proteins called “peroxins,” which are considered to be promising drug targets in pathogenic Trypanosomatidae. The first step during protein translocation across the glycosomal membrane of peroxisomal targeting signal 1 (PTS1)-harboring proteins is signal recognition by the cytosolic receptor peroxin 5 (PEX5). The C-terminal PTS1 motifs interact with the PTS1 binding domain (P1BD) of PEX5, which is made up of seven tetratricopeptide repeats. Obtaining diffraction-quality crystals of the P1BD of Trypanosoma brucei PEX5 (TbPEX5) required surface entropy reduction mutagenesis. Each of the seven tetratricopeptide repeats appears to have a residue in the α_L conformation in the loop connecting helices A and B. Five crystal structures of the P1BD of TbPEX5 were determined, each in complex with a hepta- or decapeptide corresponding to a natural or nonnatural PTS1 sequence. The PTS1 peptides are bound between the two subdomains of the P1BD. These structures indicate precise recognition of the C-terminal Leu of the PTS1 motif and important interactions between the PTS1 peptide main chain and up to five invariant Asn side chains of PEX5. The TbPEX5 structures reported here reveal a unique hydrophobic pocket in the subdomain interface that might be explored to obtain compounds that prevent relative motions of the subdomains and interfere selectively with PTS1 motif binding or release in trypanosomatids, and would therefore disrupt glycosome biogenesis and prevent parasite growth.