The inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by sterol regulatory element binding protein cleavage-activating protein requires four phenylalanine residues in span 6 of HMG-CoA reductase transmembrane domain

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway. This endoplasmic reticulum membrane protein contains a cytosolic catalytic domain and a transmembrane domain with eight membrane spans that are necessary for sterol-accelerated degradation. Competition experiments showed that wild-type transmembrane domains of HMGR and sterol regulatory element binding protein cleavage-activating protein (SCAP) blocked sterol-accelerated degradation of intact HMGR and HMGal, a model protein containing the membrane domain of HMGR linked to Escherichia coli beta-galactosidase. However, mutant transmembrane domains of HMGR and SCAP whose sterol-sensing functions were abolished did not inhibit sterol-accelerated degradation of HMGR and HMGal. In addition, our mutagenesis studies on HMGal indicated that four Phe residues conserved in span 6 of HMGR and the sterol-sensing domains of other sterol-related proteins are required for the regulated degradation of HMGR. These results suggest that HMGR and SCAP compete for binding to a sterol-regulated regulator protein, and this binding may need the four Phe residues.     

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.     

麦红吸浆虫3-羟基-3-甲基戊二酰辅酶A还原酶基因SmHMGR的克隆及在滞育与发育变态过程中的表达动态

【目的】3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是保幼激素(JH)合成途径的限速酶。麦红吸浆虫Sitodiplosis mosellana是一种典型的专性幼虫滞育昆虫。本研究旨在探讨HMGR基因在麦红吸浆虫滞育和发育变态过程中的作用。【方法】通过RT-PCR和RACE技术克隆麦红吸浆虫滞育前幼虫HMGR基因全长cDNA序列;利用生物信息学软件分析HMGR基因核苷酸和其编码的蛋白氨基酸序列特性;采用qPCR技术测定其在麦红吸浆虫滞育不同时期3龄幼虫及不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹和后蛹以及雌雄成虫)中的mRNA表达水平。【结果】克隆获得一条麦红吸浆虫HMGR基因全长cDNA序列,命名为SmHMGR(GenBank登录号: MG876766)。该基因全长2 548 bp,其中开放阅读框长2 328 bp,编码775个氨基酸,预测的蛋白分子量为84.16 kD,理论等电点为8.29。序列分析发现该基因编码的蛋白具有HMGR蛋白家族典型的HMG-CoA-reductase-classⅠ催化功能域及其他保守功能基序;序列比对和系统发育分析表明,SmHMGR与达氏按蚊Anopheles darling等长角亚目(Nematocera)昆虫HMGR的相似性最高、亲缘关系最近。SmHMGR在麦红吸浆虫滞育前的3龄早期幼虫中表达量显著升高,进入滞育后一直维持较高水平,并在滞育后静息阶段的当年12月至翌年1月达到最高。SmHMGR在蛹期表达量低于幼虫期,预蛹期表达量最低;在雌成虫中表达量显著高于在蛹和雄成虫中的表达量。【结论】SmHMGR的表达与麦红吸浆虫发育密切相关,可能在滞育诱导、维持及滞育后静息状态的维持及生殖中发挥作用,其表达量的降低可能参与了幼虫到蛹的变态。     

Bioinformatics study of the 3-hydroxy-3-methylglotaryl-coenzyme A reductase (HMGR) gene in Gramineae

Isoprenoids or terpenoids are synthesized by two important units' including dimethylallyl diphosphate and isopentenyl diphosphate (IPP). Plants use two different methods for formation of IPP, which is a cytosolic and a plastidial method. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR, EC 1.1.1.34) catalyzes the conversion of HMG-CoA to mevalonate, which is the first stage in the cytosolic pathway for biosynthesis of isoprenoid in plants. In this study, a total of fifty HMGR protein sequences from Gramineae and three animal samples including human, mouse and fruit fly were aligned and analyzed by computational tools to predict the protein properties, such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. Sequence comparison analysis revealed that there is high identity between plants and animals. Three catalytic regions including L domain, N domain and S domain were detected by structural modeling of HMGR. The tertiary structure model of Oryza sativa HMGR (Accession Number: NP_001063541) was further checked by PROCHECK algorithm, and showed that 90.3?% of the amino acid residues were located in the most favored regions in Ramachandran plot, indicating that the simulated three-dimensional structure was reliable. Phylogenetic analysis indicated that there is a relationship among species of Gramineae and other organisms. According to these results, HMGRs should be derived from a common ancestor.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) is highly associated with the cell division during the early stage of fruit development which determines the final fruit size in Litchi chinensis

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34), an enzyme catalyzing the first committed step in the mevalonic acid (MVA) pathway for the biosynthesis of isoprenoids, has been reported to be involved in the fruit size determination through the regulation of early cell division. In litchi, the cell number achieved by this early cell division determines the final fruit size, but whether HMGR plays any role in this process was unknown. In this study, we set out to address this question with gene cloning and expression analysis in fruits of different pheno- or genotypes. We found that the litchi genome includes two HMGR homologues, denoted as LcHMG1 and LcHMG2. Despite 70% sequence identity at the amino acid level, they exhibited distinct expression patterns during litchi fruit development. LcHMG1 expression was highest in the early stage of fruit development, correlated with the high level of cell division. Absolute levels of LcHMG1 expression varied among fruits of different pheno- or genotypes, with expression in large-fruited types reaching higher levels for longer duration compared to that in small-fruited types. The expression patterns for LcHMG1 strongly suggest that this gene is involved in early cell division and fruit size determination in litchi. In contrast, LcHMG2 was most highly expressed in the late stage of fruit development, in association with biosynthesis of isoprenoid compounds required for later cell enlargement. These findings provided new insights on the function of HMGR genes during fruit development.     

Computational study of conformational changes in human 3-hydroxy-3-methylglutaryl coenzyme reductase induced by substrate binding

Abstract

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR (hHMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of hHMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo hHMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of hHMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of hHMGR.

Communicated by Ramaswamy H. Sarma     

植物萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的生物信息学分析

采用生物信息学的方法和工具对已在GenBank上注册的橡胶、烟草、辣椒、穿心莲等植物的萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的核酸及氨基酸序列进行分析,并对其组成成分、信号肽、跨膜拓朴结构域、疏水性/亲水性、蛋白质二级及三级结构、分子系统进化关系等进行预测和推断。结果表明该类酶基因的全长包括5′、3′非翻译区和一个开放阅读框,无信号肽,是一个跨膜的亲水性蛋白,包括两个功能HMG-CoA结合motif及两个功能NADPH结合motif,α-螺旋和不规则盘绕是蛋白质二级结构最大量的结构元件,β-转角和延伸链散布于整个蛋白质中,蛋白质的功能域在空间布局上折叠成“V”形,“V”形的两臂由螺旋状的N结构域和S结构域构成,中间部分由L结构域构成。     

Regulation of HMG-CoA reductase degradation requires the P-type ATPase Cod1p/Spf1p

The integral ER membrane protein HMG-CoA reductase (HMGR) is a key enzyme of the mevalonate pathway from which sterols and other essential molecules are produced. HMGR degradation occurs in the ER and is regulated by mevalonate-derived signals. Little is known about the mechanisms responsible for regulating HMGR degradation. The yeast Hmg2p isozyme of HMGR undergoes regulated degradation in a manner very similar to mammalian HMGR, allowing us to isolate mutants deficient in regulating Hmg2p stability. We call these mutants cod mutants for the control of HMG-CoA reductase degradation. With this screen, we have identified the first gene of this class, COD1, which encodes a P-type ATPase and is identical to SPF1. Our data suggested that Cod1p is a calcium transporter required for regulating Hmg2p degradation. This role for Cod1p is distinctly different from that of the well-characterized Ca(2+) P-type ATPase Pmr1p which is neither required for Hmg2p degradation nor its control. The identification of Cod1p is especially intriguing in light of the role Ca(2+) plays in the regulated degradation of mammalian HMGR.     

New classification of supersecondary structures of sandwich-like proteins uncovers strict patterns of strand assemblage

To describe the supersecondary structure (SSS) of beta sandwich-like proteins (SPs), we introduce a structural unit called the "strandon." A strandon is defined as a set of sequentially consecutive strands connected by hydrogen bonds in 3D structures. Representing beta-proteins as the assembly of strandons exposes the underlying similarities in their SSS and enables us to construct a novel classification scheme of SPs. Classification of all known SPs is based on shared supersecondary structural features and is presented in the SSS database (http://binfs.umdnj.edu/sssdb/). Analysis of the SSS reveals two common specific patterns. The first pattern defines the arrangement of strandons and was found in 95% of all examined SPs. The second pattern establishes the ordering of strands in the protein domain and was observed in 82% of the analyzed SPs. Knowledge of these two patterns that uncover the spatial arrangement of strands will likely prove useful in protein structure prediction.     

Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.     

3-hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula

NORK in legumes encodes a receptor-like kinase that is required for Nod factor signaling and root nodule development. Using Medicago truncatula NORK as bait in a yeast two-hybrid assay, we identified 3-hydroxy-3-methylglutaryl CoA reductase 1 (Mt HMGR1) as a NORK interacting partner. HMGR1 belongs to a multigene family in M. truncatula, and different HMGR isoforms are key enzymes in the mevalonate biosynthetic pathway leading to the production of a diverse array of isoprenoid compounds. Testing other HMGR members revealed a specific interaction between NORK and HMGR1. Mutagenesis and deletion analysis showed that this interaction requires the cytosolic active kinase domain of NORK and the cytosolic catalytic domain of HMGR1. NORK homologs from Lotus japonicus and Sesbania rostrata also interacted with Mt HMGR1, but homologous nonsymbiotic kinases of M. truncatula did not. Pharmacological inhibition of HMGR activities decreased nodule number and delayed nodulation, supporting the importance of the mevalonate pathway in symbiotic development. Decreasing HMGR1 expression in M. truncatula transgenic roots by RNA interference led to a dramatic decrease in nodulation, confirming that HMGR1 is essential for nodule development. Recruitment of HMGR1 by NORK could be required for production of specific isoprenoid compounds, such as cytokinins, phytosteroids, or isoprenoid moieties involved in modification of signaling proteins.     

Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase.

The steady-state level of the resident endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), is regulated, in part, by accelerated degradation in response to excess sterols or mevalonate. Previous studies of a chimeric protein (HM-Gal) composed of the membrane domain of HMGR fused to Escherichia coli beta-galactosidase, as a replacement of the normal HMGR cytosolic domain, have shown that the regulated degradation of this chimeric protein, HM-Gal, is identical to that of HMGR (Chun, K. T., Bar-Nun, S., and Simoni, R. D. (1990) J. Biol. Chem. 265, 22004-22010; Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). Since the cytosolic domain can be replaced with beta-galactosidase without effect on regulated degradation, it has been assumed that the cytosolic domain was not important to this process and also that the membrane domain of HMGR was both necessary and sufficient for regulated degradation. In contrast to our previous results with HM-Gal, we observed in this study that replacement of the cytosolic domain of HMGR with various heterologous proteins can have an effect on the regulated degradation, and the effect correlates with the oligomeric state of the replacement cytosolic protein. Chimeric proteins that are oligomeric in structure are relatively stable, and those that are monomeric are unstable. To test the hypothesis that the oligomeric state of the cytosolic domain of HMGR influences degradation, we use an "inducible" system for altering the oligomeric state of a protein in vivo. Using a chimeric protein that contains the membrane domain of HMGR fused to three copies of FK506-binding protein 12, we were able to induce oligomerization by addition of a "double-headed" FK506-like "dimerizer" drug (AP1510) and to monitor the degradation rate of both the monomeric form and the drug-induced oligomeric form of the protein. We show that this chimeric protein, HM-3FKBP, is unstable in the monomeric state and is stabilized by AP1510-induced oligomerization. We also examined the degradation rate of HMGR as a function of concentrations within the cell. HMGR is a functional dimer; therefore, its oligomeric state and, we predict, its degradation rate should be concentration-dependent. We observed that it is degraded more rapidly at lower concentrations.     

A novel lysine-rich domain and GTP binding motifs regulate the nucleolar retention of human guanine nucleotide binding protein, GNL3L

A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1-50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus.     

The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3′ extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3′ end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.     

Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment

TPST1 is a human tyrosylprotein sulfotransferase that uses 3'phosphoadenosine-5'phosphosulfate (PAPS) to transfer the sulfate moiety to proteins predominantly designated for secretion. To achieve a general understanding of the cellular role of human tyrosine-directed sulfotransferases, we investigated targeting, structure and posttranslational modification of TPST1. Golgi localisation of the enzyme in COS-7 and HeLa cells was visualised by fluorescence imaging techniques. PNGase treatment and mutational studies determined that TPST1 bears N-linked glycosyl residues exclusively at position Asn60 and Asn262. By alanine mutation of these asparagine residues, we could determine that the N-linked oligosaccharides do not have an influence on Golgi retention of TPST1. In concert with N and C-terminal flanking residues, the transmembrane domain of TPST1 was determined to act in targeting and retention of the enzyme to the trans-Golgi compartment. This domain exhibits a pronounced secondary structure in a lipid environment. Further in vivo FRET studies using the transmembrane domain suggest that the human tyrosylprotein sulfotransferase may be functional as homodimer/oligomer in the trans-Golgi compartment.     

The structure of a tandem pair of spectrin repeats of plectin reveals a modular organization of the plakin domain

Plectin is a large and versatile cytoskeletal linker and member of the plakin protein family. Plakins share a conserved region called the plakin domain located near their N terminus. We have determined the crystal structure of an N-terminal fragment of the plakin domain of plectin to 2.05 A resolution. This region is adjacent to the actin-binding domain and is required for efficient binding to the integrin alpha6beta4 in hemidesmosomes. The structure is formed by two spectrin repeats connected by an alpha-helix that spans these two repeats. While the first repeat is very similar to other known structures, the second repeat is structurally different with a hydrophobic core, narrower than that in canonical spectrin repeats. Sequence analysis of the plakin domain revealed the presence of up to nine consecutive spectrin repeats organized in an array of tandem modules, and a Src-homology 3 domain inserted in the central spectrin repeat. The structure of the plakin domain is reminiscent of the modular organization of members of the spectrin family. The architecture of the plakin domain suggests that it forms an elongated and flexible structure, and provides a novel molecular explanation for the contribution of plectin and other plakins to the elasticity and stability of tissues subjected to mechanical stress, such as the skin and striated muscle.     

细纹豆芫菁3-羟甲基戊二酰辅酶A-还原酶基因全长cDNA克隆及生物信息学分析

3-羟甲基戊二酰辅酶A-还原酶（3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR）是甲羟戊酸途径的关键酶。获得芫菁体内HMGR基因信息是确定甲羟戊酸途径与斑蝥素合成相关性的基础。本研究利用RACE技术从细纹豆芫菁Epicauta mannerheimi (Maklin)体内克隆获得HMGR基因全长cDNA序列, 命名为EmHMGR（GenBank登录号为JQ690539）。该基因全长3 118 bp, 其中5′端非翻译区178 bp, 3′端非翻译区414 bp, 开放阅读框2 526 bp, 编码842个氨基酸。推测的蛋白质分子量为92.8 kDa, 理论等电点为6.0, 预测分子式为C₄₁₃₅H₆₆₀₄N1₀₉₈O₁₂₁₆S₅₀, 不稳定系数为43.37, 总亲水性系数为0.091, 为疏水性不稳定蛋白。序列分析发现该基因编码的蛋白与已报道的其他昆虫HMGR的氨基酸序列一致性达50%以上, 而且包含HMGR_Class I保守功能域、 固醇敏感多肽区及HMGR蛋白的其他保守功能位点。系统进化分析发现该基因与叶甲科昆虫HMGR基因的关系最近。本研究首次从芫菁科昆虫体内克隆获得甲羟戊酸途径的关键酶EmHMGR基因, 为后期芫菁体内斑蝥素生物合成途径的研究奠定了基础。     

Molecular Determinants of PAM2 Recognition by the MLLE Domain of Poly(A)-Binding Protein

MLLE (previously known as PABC) is a peptide-binding domain that is found in poly(A)-binding protein (PABP) and EDD (E3 isolated by differential display), a HECT E3 ubiquitin ligase also known as HYD (hyperplastic discs tumor suppressor) or UBR5. The MLLE domain from PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12 amino acid peptide motif called PAM2 (for PABP-interacting motif 2). Here, we determined crystal structures of the MLLE domain from PABP alone and in complex with PAM2 peptides from PABP-interacting protein 2. The structures provide a detailed view of hydrophobic determinants of the MLLE binding coded by PAM2 positions 3, 5, 7, 10, and 12 and reveal novel intermolecular polar contacts. In particular, the side chain of the invariant MLLE residue K580 forms hydrogen bonds with the backbone of PAM2 residues 5 and 7. The structures also show that peptide residues outside of the conserved PAM2 motif contribute to binding. Altogether, the structures provide a significant advance in understanding the molecular basis for the binding of PABP by PAM2-containing proteins involved in translational control, mRNA deadenylation, and other cellular processes.