Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif

The eukaryotic DNA replication protein Mcm10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) Mcm10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase α is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMcm10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to Mcm10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate Mcm10. Our results provide insight in the action of Mcm10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.     

Structure of the Toxoplasma gondii ROP18 Kinase Domain Reveals a Second Ligand Binding Pocket Required for Acute Virulence

At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence.     

Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin β-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)₂GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.Many transmembrane signaling systems consist of specific G protein-coupled receptors (GPCRs)³ that transduce a diverse array of extracellular stimuli into intracellular signaling events (1). GPCRs modulate the activity of numerous effector molecules and regulate multiple biological functions including neurotransmission, sensory perception, cardiovascular function, development, and cell growth and differentiation (2). To ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and duration, these signaling cascades are tightly regulated. GPCRs are subject to three principle modes of regulation; 1) desensitization, in which a receptor becomes refractory to continued stimuli; 2) endocytosis, where receptors are removed from the cell surface; 3) down-regulation, where total receptor levels are decreased (3, 4). Agonist-dependent regulation is primarily mediated by GPCR kinases that specifically phosphorylate activated GPCRs and initiate the recruitment of arrestins. Arrestins are divided into two major classes, visual and non-visual, based on their localization and function. The non-visual arrestins, arrestin2 and 3 (also termed β-arrestin1 and -2, respectively), are broadly distributed and function in multiple processes including GPCR desensitization, trafficking, and signaling (4–6).Initial structural insight on arrestins was provided by the x-ray crystal structure of bovine arrestin1 (7, 8), whereas the crystal structures of C-terminal-truncated (9) and wild type (10) bovine arrestin2 and salamander arrestin4 (11) have also been solved. In general, arrestins are composed of two major domains made up of β strands and connecting loops that are held together by a polar core region consisting of buried salt bridges. It has been proposed that arrestins adopt an active conformation upon binding to phosphorylated receptors, which disrupts the polar core resulting in the release of the C-terminal tail (12). Disruption of the polar core by point mutation of Arg-169 generates a constitutively active arrestin2, which mimics the active state. This mutated arrestin binds to the β₂-adrenergic receptor (β₂AR) in a phosphorylation-independent manner, induces internalization of a δ-opioid receptor lacking phosphorylation sites (13), and has increased binding to clathrin and AP-2 (14).A role for non-visual arrestins in GPCR endocytosis was first described for the β₂AR (15, 16), although it is now evident that arrestins regulate the trafficking of multiple GPCRs as well as additional classes of receptors (4). An early step in this process involves arrestin binding to an activated phosphorylated receptor that enhances arrestin interaction with the endocytic proteins, clathrin, and AP-2 (16, 17). An additional important step in this process involves arrestin interaction with phosphoinositides such as phosphatidylinositol diphosphate and trisphosphate (18). Although the dynamics of these interactions have not been studied, arrestin2 and -3 have been shown to interact specifically and stoichiometrically with clathrin (16). Furthermore, fluorescence microscopy reveals that activated β₂AR, arrestin2, clathrin, and AP-2 all colocalize upon receptor stimulation (16). The primary clathrin binding determinant in arrestin2, LIELD, spans residues 376–380 and is located in an extended disordered loop that immediately precedes the final C-terminal β-strand (10, 19). This region, the clathrin binding box, is consistent with a consensus motif, LϕXϕ(D/E) (where ϕ is a bulky hydrophobic residue, and X represents any polar amino acid), established in other clathrin-binding proteins including AP-2 (20), AP180 (21), amphiphysin (22), and epsin (23). Importantly, the mutation of this motif in arrestin3 and its deletion in arrestin2 significantly disrupts clathrin binding and receptor endocytosis (14, 19). A mutagenesis study of clathrin localized an arrestin binding site to the N-terminal domain of the clathrin heavy chain, specifically residues Glu-89, Lys-96, and Lys-98 (24). Moreover, a crystal structure of clathrin-(1–363) in complex with an arrestin3 peptide (residues 369–381) supports the mutagenesis data and the predicted location of the arrestin-clathrin interaction site (25).To further elucidate the mechanisms involved in mediating arrestin/clathrin interaction, we have determined the crystal structure of clathrin with the short (arrestin2S) and long (arrestin2L) isoforms of arrestin2, which differ by an 8-amino acid insert between β strands 18 and 19 (26). Our results identify an additional and unique interaction encoded in the arrestin2L isoform that is distinct from the previously well characterized interaction involving the LϕXϕ(D/E) motif. Specifically, we observe that the 8 amino acid splice loop in arrestin2L interacts with a pocket formed by blades 4 and 5 in clathrin. Biochemical and cell biological analysis confirm a role for both binding sites in arrestin2L/clathrin interaction and demonstrate an essential role of these interactions in arrestin-mediated GPCR endocytosis.     

Structure of Osh3 Reveals a Conserved Mode of Phosphoinositide Binding in Oxysterol-Binding Proteins

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The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.     

Crystal Structures of the Kinase Domain of the Sulfate-Activating Complex in Mycobacterium tuberculosis

In Mycobacterium tuberculosis the sulfate activating complex provides a key branching point in sulfate assimilation. The complex consists of two polypeptide chains, CysD and CysN. CysD is an ATP sulfurylase that, with the energy provided by the GTPase activity of CysN, forms adenosine-5’-phosphosulfate (APS) which can then enter the reductive branch of sulfate assimilation leading to the biosynthesis of cysteine. The CysN polypeptide chain also contains an APS kinase domain (CysC) that phosphorylates APS leading to 3’-phosphoadenosine-5’-phosphosulfate, the sulfate donor in the synthesis of sulfolipids. We have determined the crystal structures of CysC from M. tuberculosis as a binary complex with ADP, and as ternary complexes with ADP and APS and the ATP mimic AMP-PNP and APS, respectively, to resolutions of 1.5 Å, 2.1 Å and 1.7 Å, respectively. CysC shows the typical APS kinase fold, and the structures provide comprehensive views of the catalytic machinery, conserved in this enzyme family. Comparison to the structure of the human homolog show highly conserved APS and ATP binding sites, questioning the feasibility of the design of specific inhibitors of mycobacterial CysC. Residue Cys556 is part of the flexible lid region that closes off the active site upon substrate binding. Mutational analysis revealed this residue as one of the determinants controlling lid closure and hence binding of the nucleotide substrate.     

胰岛素受体底物1的PH结构域与PKC的结合

从大鼠的肝脏克隆胰岛素受体底物１（ＩＲＳ－１）的ＰＨ结构域基因并进行谷胱甘肽Ｓ－转移酶（ＧＳＴ）融合表达,研究该结构域与蛋白激酶Ｃ（ＰＫＣ）的结合情况,并为进一步寻找其新配基打下基础,研究采用一步法从大鼠新鲜肝组织中提取总ＲＮＡ,以ＲＴ－ＰＣＲ的方法扩增目的基因片段,测序证明序列正确,再将正确的目的基因片段定向克隆到表达载体ｐＧＥＸ－４Ｔ－１中,以ＩＰＴＧ在２６℃下诱导,获得与ＧＳＴ的融合表达,表     

Ligand Binding in the Conserved Interhelical Loop of CorA, a Magnesium Transporter from Mycobacterium tuberculosis

CorA is a constitutively expressed magnesium transporter in many bacteria. The crystal structures of Thermotoga maritima CorA provide an excellent structural framework for continuing studies. Here, the ligand binding properties of the conserved interhelical loop, the only portion of the protein exposed to the periplasmic space, are characterized by solution nuclear magnetic resonance spectroscopy. Through titration experiments performed on the isolated transmembrane domain of Mycobacterium tuberculosis CorA, it was found that two CorA substrates (Mg²⁺ and Co²⁺) and the CorA-specific inhibitor (Co(III) hexamine chloride) bind in the loop at the same binding site. This site includes the glutamic acid residue from the conserved “MPEL” motif. The relatively large dissociation constants indicate that such interactions are weak but not atypical for channels. The present data support the hypothesis that the negatively charged loop could act as an electrostatic ring, increasing local substrate concentrations before transport across the membrane.The heterogeneous membrane environment is very challenging to mimic for structural, dynamic, and functional studies of membrane proteins. It is not surprising, therefore, that different aspects of the structure can be brought to light under different conditions. Recently, an excellent set of crystal structures of the CorA Mg²⁺ transporter have been published (1–3), and whereas many CorA mysteries were solved, the highly conserved periplasmic interhelical loops in the pentameric structure were not well resolved. Here, solution nuclear magnetic resonance (NMR) spectroscopy of the transmembrane domain resolves these loops, and the secondary structure and ion binding in this domain are characterized.The 2TM-GxN family of transporters is a large group of integral membrane proteins responsible for metal ion transport (especially for magnesium) across membranes (4, 5). CorA is a prototypical member in this family responsible for magnesium influx as well as efflux in some cases (5, 6). In the extensive phylogenetic analysis, it was shown that CorA is characterized by a universally conserved “GMN” motif in an interhelical loop connecting two conserved transmembrane helices at the C terminus of the full-length protein (4). As the only constitutively expressed magnesium transporter, CorA can play an important role in the viability of pathogens, such as Helicobacter pylori (7).Pentameric CorA from Thermotoga maritima forms two distinguishable domains; that is, a large cytoplasmic domain and a small transmembrane domain (1–3). In this latter domain there are two transmembrane helices connected by an interhelical periplasmic loop. The first transmembrane helix (TM1)2 lines the pore, whereas the second transmembrane helix (TM2) forms an outer ring of helices, which appears to have only weak interactions with the TM1 helices.Different mechanisms of substrate transport for CorA have been proposed (1–3, 6, 8), whereas the structure and function of the conserved loop is still an open issue. Based on phylogenetic analyses, it has been shown that there were two conserved sequences in the interhelical loop. One is the GMN motif that is universally conserved, and the other is the MPEL motif that is conserved throughout most bacterial genomes. The glutamic acid residue in the MPEL sequence is almost universally conserved in CorA and CorA homologs in eukaryotic cells, including yeast and humans (4). However, it is not conserved in Methanococcus jannaschii for which CorA has been functionally characterized (9). Although the Mycobacterium tuberculosis protein, studied here, has not been functionally characterized in detail, it has been shown to transport magnesium ions across the membrane.³ Because the interhelical loop is the only portion of the protein exposed to the periplasmic side and because there is a highly conserved negatively charged residue, it has been suggested that the loop could act as an initial magnesium binding site (1). This could result in enhancing Mg²⁺ concentration at the mouth of the pore, enhancing substrate selection and generating partial cation dehydration (1). Recent functional studies of CorA homologs from yeast have shown that this negatively charged residue in the loop plays an important role in function (10–12). Substitution of Glu by Lys results in a dramatic reduction in transport activity in yeast (10), and substitution of a positively charged residue (Arg in A1r2p, a CorA homolog in yeast) by a Glu increases channel activity (11). However, the CorA from M. jannaschii does not contain either this residue or a negatively charged loop. Based on these results, the negatively charged loop appears to be functionally important and may act as an initial substrate binding site for increasing the local substrate concentration to facilitate ion transport (10–12), whereas it may not be functionally essential at least for some CorA members. However, it has also been argued, based on limited crystallographic data, that the loop may not form a binding site for substrate selection and dehydration (3). Instead, it was speculated that the loop could mediate the relative movement of the two transmembrane helices (3). Accordingly, further investigation of this functionally important loop is warranted.In the present work we characterize the interaction between the isolated transmembrane domain of CorA (CorA-TMD) and its substrates as well as an inhibitor. Such a “divide and conquer” strategy has been successfully applied to several membrane proteins, such as the M2 protein (a proton channel from influenza A) (13–15), Vpu (a membrane protein encoded by human immunodeficiency virus involved in the budding of new viral particles from the host cell) (16, 17), GlpG (a rhomboid intra-membrane protease) (18, 19), and S2P (a intra-membrane metalloprotease) (20). In addition, the electron density map from the crystal structure of the isolated CorA soluble domain was used to solve the structure of full-length CorA by molecular replacement (1). This suggests that the structural influence of the transmembrane domain on the soluble domain is limited, at least for the conformational state that was crystallized (1). Hence, a divide and conquer strategy for CorA appears to be justified and provides an opportunity to characterize this domain by solution NMR.It has been shown that NMR has a unique ability of characterize weak interactions that are not readily characterized by other methods (21, 22). In the present work we characterize the binding of two substrates (Mg²⁺ and Co²⁺) to the loop as well as an inhibitor (Cobalt(III) hexamine chloride, HexCo³⁺) by NMR. Our data indicate that the ligands of CorA can weakly but specifically bind to the interhelical loop of CorA-TMD through their interaction with the conserved negatively charged glutamic acid residue in the MPEL motif, supporting the hypothesis that the loop acts as an initial binding site.     

Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase

PknH Ser/Thr protein kinase of Mycobacterium tuberculosis controls the expression of a variety of cell wall related enzymes and regulates the in vivo growth in mice. Therefore, we predicted that the PknH kinase could phosphorylate several substrates controlling different metabolic and physiological pathways. Using a bioinformatic approach, we identified 40 potential substrates. Two substrates were shown to be phosphorylated by recombinant PknH kinase in vitro. Point mutation studies verified that substrates are phosphorylated at the in silico-predicted sites. Kinetic studies revealed a similar relative-phosphorylation rate (V(max)) of PknH towards two new substrates and the only previously known substrate, EmbR. Unlike the EmbR protein, the Rv0681 and DacB1 proteins do not contain an FHA domain and are possible participants of new signaling pathways mediated by the PknH kinase in M. tuberculosis.     

Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 Å resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.     

Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB

LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal α-helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp²⁵) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves.     

Conformational Coupling between Receptor and Kinase Binding Sites through a Conserved Salt Bridge in a Signaling Complex Scaffold Protein

Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD), NMR spectroscopy, and circular dichroism (CD), we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.     

Crystal Structure of the Mineralocorticoid Receptor DNA Binding Domain in Complex with DNA

The steroid hormone receptors regulate important physiological functions such as reproduction, metabolism, immunity, and electrolyte balance. Mutations within steroid receptors result in endocrine disorders and can often drive cancer formation and progression. Despite the conserved three-dimensional structure shared among members of the steroid receptor family and their overlapping DNA binding preference, activation of individual steroid receptors drive unique effects on gene expression. Here, we present the first structure of the human mineralocorticoid receptor DNA binding domain, in complex with a canonical DNA response element. The overall structure is similar to the glucocorticoid receptor DNA binding domain, but small changes in the mode of DNA binding and lever arm conformation may begin to explain the differential effects on gene regulation by the mineralocorticoid and glucocorticoid receptors. In addition, we explore the structural effects of mineralocorticoid receptor DNA binding domain mutations found in type I pseudohypoaldosteronism and multiple types of cancer.     

Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch

TbBILBO1 is the only known component of the flagellar pocket collar, a cytoskeletal barrier element found in trypanosomes. The N-terminal domain (NTD) of TbBILBO1 was found to be dispensable for targeting of the protein in vivo. However, overexpression of constructs lacking the NTD caused complete growth inhibition, implying an essential requirement for this domain. A high resolution structure of the NTD of TbBILBO1 showed that it forms a ubiquitin-like fold with a conserved surface patch. Mutagenesis of this patch recapitulated the phenotypic effects of deleting the entire domain and was found to cause cell death. The surface patch on the NTD of TbBILBO1 is therefore a potential drug target.     

ErbB2受体酪氨酸激酶激酶区的表达与纯化

以GFP融合表达的形式在毕赤酵母中表达具有生物活性的受体酪氨酸激酶ErbB2的激酶区.构建受体酪氨酸激酶激酶区与GFP的融合表达载体pPIC3.5K,转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G418高拷贝菌株筛选,以及摇瓶诱导表达筛选,选取较高水平表达菌株进行5升罐培养,以镍亲和层析手段纯化得到蛋白表达产物,进行SDS-PAGE分析和酶联免疫反应检测酶活.结果表明在毕赤酵母中成功诱导表达了约100kD的激酶融合蛋白并具有激酶活性.该研究为筛选ErbB2的抑制剂奠定了基础.