Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 31
Filter
1.
Nature ; 550(7677): 534-538, 2017 10 26.
Article in English | MEDLINE | ID: mdl-29045385

ABSTRACT

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.


Subject(s)
Aminopyridines/chemistry , Aminopyridines/pharmacology , Indazoles/chemistry , Indazoles/pharmacology , Phenols/chemistry , Phenols/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Ubiquitin/metabolism , Animals , Binding, Competitive , Cell Line, Tumor , Drug Synergism , Female , Humans , Mice , Mice, SCID , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/pathology , Protein Binding , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Substrate Specificity , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin/chemistry , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/deficiency , Ubiquitin-Specific Peptidase 7/metabolism
2.
J Chem Inf Model ; 62(3): 472-485, 2022 02 14.
Article in English | MEDLINE | ID: mdl-35029985

ABSTRACT

Nuclear magnetic resonance (NMR) data from NOESY (nuclear Overhauser enhancement spectroscopy) and ROESY (rotating frame Overhauser enhancement spectroscopy) experiments can easily be combined with distance geometry (DG) based conformer generators by modifying the molecular distance bounds matrix. In this work, we extend the modern DG based conformer generator ETKDG, which has been shown to reproduce experimental crystal structures from small molecules to large macrocycles well, to include NOE-derived interproton distances. In noeETKDG, the experimentally derived interproton distances are incorporated into the distance bounds matrix as loose upper (or lower) bounds to generate large conformer sets. Various subselection techniques can subsequently be applied to yield a conformer bundle that best reproduces the NOE data. The approach is benchmarked using a set of 24 (mostly) cyclic peptides for which NOE-derived distances as well as reference solution structures obtained by other software are available. With respect to other packages currently available, the advantages of noeETKDG are its speed and that no prior force-field parametrization is required, which is especially useful for peptides with unnatural amino acids. The resulting conformer bundles can be further processed with the use of structural refinement techniques to improve the modeling of the intramolecular nonbonded interactions. The noeETKDG code is released as a fully open-source software package available at www.github.com/rinikerlab/customETKDG.


Subject(s)
Peptides, Cyclic , Peptides , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Protein Conformation
3.
J Biol Chem ; 291(31): 16292-306, 2016 07 29.
Article in English | MEDLINE | ID: mdl-27226599

ABSTRACT

Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/chemistry , Microtubule-Associated Proteins/chemistry , Neuropeptides/chemistry , Single-Chain Antibodies/chemistry , Animals , Camelus , Cryoelectron Microscopy , Crystallography, X-Ray , Doublecortin Domain Proteins , Humans , Mice , Protein Domains , Protein Structure, Quaternary , Rabbits
4.
Bioorg Med Chem Lett ; 27(3): 632-635, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28025004

ABSTRACT

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26pmol/mg protein.


Subject(s)
Protein Kinase Inhibitors/chemistry , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyridones/chemistry , Agammaglobulinaemia Tyrosine Kinase , Glutathione/chemistry , Magnetic Resonance Spectroscopy , Microsomes/metabolism , Protein Kinase Inhibitors/metabolism , Protein-Tyrosine Kinases/metabolism , Pyridones/metabolism , Tandem Mass Spectrometry
5.
J Biol Chem ; 289(2): 942-55, 2014 Jan 10.
Article in English | MEDLINE | ID: mdl-24225950

ABSTRACT

PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 µm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 µm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the ß-strand and a discontinuous short α-helix, and it engaged in the same ß-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.


Subject(s)
Oligopeptides/pharmacology , Proprotein Convertases/metabolism , Receptors, LDL/metabolism , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive/drug effects , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hep G2 Cells , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/metabolism , Peptide Library , Proprotein Convertase 9 , Proprotein Convertases/chemistry , Proprotein Convertases/genetics , Protein Binding/drug effects , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, LDL/chemistry , Receptors, LDL/genetics , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics
6.
Proc Natl Acad Sci U S A ; 109(29): 11788-93, 2012 Jul 17.
Article in English | MEDLINE | ID: mdl-22745160

ABSTRACT

Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo- and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMX-overexpressing cancers.


Subject(s)
Apoptosis/physiology , Hydantoins/pharmacology , Models, Molecular , Nuclear Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Blotting, Western , Cell Cycle Proteins , Cell Line, Tumor , Crystallization , Dimerization , Fluorescence Resonance Energy Transfer , Humans , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/chemistry , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-mdm2/chemistry , Signal Transduction/drug effects , Signal Transduction/physiology , Tetrazolium Salts , Thiazoles
7.
Proc Natl Acad Sci U S A ; 108(26): 10484-9, 2011 Jun 28.
Article in English | MEDLINE | ID: mdl-21670263

ABSTRACT

Erythroid Krüppel-like factor (EKLF) plays an important role in erythroid development by stimulating ß-globin gene expression. We have examined the details by which the minimal transactivation domain (TAD) of EKLF (EKLFTAD) interacts with several transcriptional regulatory factors. We report that EKLFTAD displays homology to the p53TAD and, like the p53TAD, can be divided into two functional subdomains (EKLFTAD1 and EKLFTAD2). Based on sequence analysis, we found that EKLFTAD2 is conserved in KLF2, KLF4, KLF5, and KLF15. In addition, we demonstrate that EKLFTAD2 binds the amino-terminal PH domain of the Tfb1/p62 subunit of TFIIH (Tfb1PH/p62PH) and four domains of CREB-binding protein/p300. The solution structure of the EKLFTAD2/Tfb1PH complex indicates that EKLFTAD2 binds Tfb1PH in an extended conformation, which is in contrast to the α-helical conformation seen for p53TAD2 in complex with Tfb1PH. These studies provide detailed mechanistic information into EKLFTAD functions as well as insights into potential interactions of the TADs of other KLF proteins. In addition, they suggest that not only have acidic TADs evolved so that they bind using different conformations on a common target, but that transitioning from a disordered to a more ordered state is not a requirement for their ability to bind multiple partners.


Subject(s)
Kruppel-Like Transcription Factors/chemistry , Amino Acid Sequence , Binding Sites , Calorimetry , Cloning, Molecular , Humans , K562 Cells , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Transcription Factors/metabolism
8.
Nat Cancer ; 4(6): 812-828, 2023 06.
Article in English | MEDLINE | ID: mdl-37277530

ABSTRACT

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.


Subject(s)
Neoplasms , Proto-Oncogene Proteins p21(ras) , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Precision Medicine , Transcription Factors/metabolism , Signal Transduction
9.
J Virol ; 85(11): 5287-300, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21450828

ABSTRACT

The papillomavirus E1 helicase, with the help of E2, assembles at the viral origin into a double hexamer that orchestrates replication of the viral genome. The N-terminal region (NTR) of E1 is essential for DNA replication in vivo but dispensable in vitro, suggesting that it has a regulatory function. By deletion analysis, we identified a conserved region of the E1 NTR needed for efficient replication of viral DNA. This region is predicted to form an amphipathic α-helix (AH) and shows sequence similarity to portions of the p53 and herpes simplex virus (HSV) VP16 transactivation domains known as transactivation domain 2 (TAD2) and VP16C, which fold into α-helices upon binding their target proteins, including the Tfb1/p62 (Saccharomyces cerevisiae/human) subunit of general transcription factor TFIIH. By nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we found that a peptide spanning the E1 AH binds Tfb1 on the same surface as TAD2/VP16C and with a comparable affinity, suggesting that it does bind as an α-helix. Furthermore, the E1 NTRs from several human papillomavirus (HPV) types could activate transcription in yeast, and to a lesser extent in mammalian cells, when fused to a heterologous DNA-binding domain. Mutation of the three conserved hydrophobic residues in the E1 AH, analogous to those in TAD2/VP16C that directly contact their target proteins, decreased transactivation activity and, importantly, also reduced by 50% the ability of E1 to support transient replication of DNA in C33A cells, at a step following assembly of the E1-E2-ori preinitiation complex. These results demonstrate the existence of a conserved TAD2/VP16C-like AH in E1 that is required for efficient replication of viral DNA.


Subject(s)
DNA Helicases/metabolism , DNA, Viral/metabolism , Papillomaviridae/physiology , Trans-Activators/metabolism , Viral Proteins/metabolism , Virus Replication , Calorimetry , DNA Helicases/genetics , DNA Mutational Analysis , Epithelial Cells/virology , Humans , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Secondary , Saccharomyces cerevisiae/genetics , Sequence Deletion , Sequence Homology, Amino Acid , Trans-Activators/genetics , Viral Proteins/genetics
10.
Proc Natl Acad Sci U S A ; 106(18): 7408-13, 2009 May 05.
Article in English | MEDLINE | ID: mdl-19376969

ABSTRACT

Several peptide hormones are initially synthesized as inactive precursors. It is only on entry of these prohormones and their processing proteases into dense core secretory granules (DCSGs) that the precursors are cleaved to generate their active forms. Prohormone convertase (PC)1/3 is a processing protease that is targeted to DCSGs. The signal for targeting PC1/3 to DCSGs resides in its carboxy-terminal tail (PC1/3(617-753)), where 3 regions (PC1/3(617-625), PC1/3(665-682), and PC1/3(711-753)) are known to aid in sorting and membrane association. In this article, we have determined a high-resolution structure of the extreme carboxy-terminal sorting domain, PC1/3(711-753) in micelles by NMR spectroscopy. PC1/3(711-753) contains 2 alpha helices located between residues 722-728 and 738-750. Functional assays demonstrate that the second helix (PC1/3(738-750)) is necessary and sufficient to target a constitutively secreted protein to granules, and that L(745) anchors a hydrophobic patch that is critical for sorting. Also, we demonstrate that calcium binding by the second helix of PC1/3(711-753) promotes aggregation of the domain via the hydrophobic patch centered on L(745). These results provide a structure-function analysis of a DCSG-sorting domain, and reveal the importance of a hydrophobic patch and calcium binding in controlling the sorting of proteins containing alpha helices to DCSGs.


Subject(s)
Proprotein Convertase 1/chemistry , Secretory Vesicles/enzymology , Animals , Calcium/chemistry , Mice , Nuclear Magnetic Resonance, Biomolecular , Proprotein Convertase 1/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport
11.
Blood ; 114(1): 165-73, 2009 Jul 02.
Article in English | MEDLINE | ID: mdl-19411634

ABSTRACT

In addition to orchestrating the expression of all erythroid-specific genes, GATA-1 controls the growth, differentiation, and survival of the erythroid lineage through the regulation of genes that manipulate the cell cycle and apoptosis. The stages of mammalian erythropoiesis include global gene inactivation, nuclear condensation, and enucleation to yield circulating erythrocytes, and some of the genes whose expression are altered by GATA-1 during this process are members of the p53 pathway. In this study, we demonstrate a specific in vitro interaction between the transactivation domain of p53 (p53TAD) and a segment of the GATA-1 DNA-binding domain that includes the carboxyl-terminal zinc-finger domain. We also show by immunoprecipitation that the native GATA-1 and p53 interact in erythroid cells and that activation of p53-responsive promoters in an erythroid cell line can be inhibited by the overexpression of GATA-1. Mutational analysis reveals that GATA-1 inhibition of p53 minimally requires the segment of the GATA-1 DNA-binding domain that interacts with p53TAD. This inhibition is reciprocal, as the activation of a GATA-1-responsive promoter can be inhibited by p53. Based on these findings, we conclude that inhibition of the p53 pathway by GATA-1 may be essential for erythroid cell development and survival.


Subject(s)
GATA1 Transcription Factor/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Animals , Binding Sites , Cell Differentiation , Cell Line, Tumor , Cell Survival , DNA/chemistry , DNA/metabolism , Erythroid Cells/cytology , Erythroid Cells/metabolism , GATA1 Transcription Factor/chemistry , GATA1 Transcription Factor/genetics , Humans , In Vitro Techniques , Macromolecular Substances , Mice , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Zinc Fingers
12.
Proc Natl Acad Sci U S A ; 105(1): 106-11, 2008 Jan 08.
Article in English | MEDLINE | ID: mdl-18160537

ABSTRACT

The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the alpha-subunit of the general transcription factor IIE (TFIIEalpha). We have determined that the acidic carboxyl terminus of TFIIEalpha (TFIIEalpha(336-439)) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEalpha binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEalpha(336-439) is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEalpha(336-439) and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEalpha(336-439) contains a small domain (residues 395-433) folded in a novel betabetaalphaalphaalpha topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for TFIIEalpha(336-439) binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEalpha(336-439) can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEalpha and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Mitochondrial Proteins/metabolism , Transcription Factor TFIIH/metabolism , Transcription Factors, TFII/metabolism , Transcription Factors/metabolism , Tumor Suppressor Protein p53/physiology , Binding Sites , Calorimetry/methods , Humans , Kinetics , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Mutagenesis , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Transcription Factors, TFII/chemistry , Tumor Suppressor Protein p53/metabolism
13.
Biochemistry ; 48(9): 1964-74, 2009 Mar 10.
Article in English | MEDLINE | ID: mdl-19215094

ABSTRACT

Recycling of RNA polymerase II (RNAPII) requires dephosphorylation of the C-terminal domain (CTD) of the largest subunit of the polymerase. FCP1 enables the recycling of RNAPII via its CTD-specific phosphatase activity, which is stimulated by the RAP74 subunit of the general transcription factor TFIIF. Both the central (centFCP1) and C-terminal (cterFCP1) domains of FCP1 interact independently and specifically with the C-terminal domain of RAP74 (cterRAP74), suggesting that these interactions mediate the stimulatory effect of TFIIF on the CTD phosphatase activity of FCP1. Phosphorylation of FCP1 by casein kinase 2 on residues in its central (T584) and C-terminal (S942 and S944) domains stimulates its binding to RAP74 and its CTD phosphatase activity. To improve our understanding of the FCP1-RAP74 interactions, we previously determined the NMR structure of a complex formed by human cterRAP74 and cterFCP1. We now present the high-resolution NMR structure and thermodynamic characterization by isothermal titration calorimetry of a complex formed by the same cterRAP74 domain and a phosphorylated peptide from the central domain of human FCP1 (centFCP1-PO(4)). Comparison of the cterFCP1-cterRAP74 and centFCP1-PO(4)-cterRAP74 complexes indicates that centFCP1 and cterFCP1 both utilize hydrophobic and acidic residues to recognize the same groove of RAP74, but there are significant differences in the details of their interactions. These differences point to the adaptability of RAP74 to recognize the two regions of FCP1. Our NMR and thermodynamic studies further elucidate the complex molecular mechanism by which TFIIF and FCP1 cooperate for RNAPII recycling.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Phosphoprotein Phosphatases/chemistry , Transcription Factors, TFII/chemistry , Amino Acid Sequence , Binding Sites , Humans , Molecular Sequence Data , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , RNA Polymerase II/metabolism , Sequence Homology, Amino Acid , Serine/chemistry , Serine/metabolism , Structure-Activity Relationship , Thermodynamics , Threonine/chemistry , Threonine/metabolism , Transcription Factors, TFII/genetics , Transcription Factors, TFII/metabolism
14.
J Am Chem Soc ; 130(32): 10596-604, 2008 Aug 13.
Article in English | MEDLINE | ID: mdl-18630911

ABSTRACT

The Herpes Simplex Virion Protein 16 (VP16) activates transcription through a series of protein/protein interactions involving its highly acidic transactivation domain (TAD). The acidic TAD of VP16 (VP16TAD) has been shown to interact with several partner proteins both in vitro and in vivo, and many of these VP16 partners also bind the acidic TAD of the mammalian tumor suppressor protein p53. For example, the TADs of VP16 and p53 (p53TAD) both interact directly with the p62/Tfb1 (human/yeast) subunit of TFIIH, and this interaction correlates with their ability to activate both the initiation and elongation phase of transcription. In this manuscript, we use NMR spectroscopy, isothermal titration calorimetery (ITC) and site-directed mutagenesis studies to characterize the interaction between the VP16TAD and Tfb1. We identify a region within the carboxyl-terminal subdomain of the VP16TAD (VP16C) that has sequence similarity with p53TAD2 and binds Tfb1 with nanomolar affinity. We determine an NMR structure of a Tfb1/VP16C complex, which represents the first high-resolution structure of the VP16TAD in complex with a target protein. The structure demonstrates that like p53TAD2, VP16C forms a 9-residue alpha-helix in complex with Tfb1. Comparison of the VP16/Tfb1and p53/Tfb1 structures clearly demonstrates how the viral activator VP16C and p53TAD2 shares numerous aspects of binding to Tfb1. Despite the similarities, important differences are observed between the p53TAD2/Tfb1 and VP16C/Tfb1 complexes, and these differences demonstrate how selected activators such as p53 depend on phosphorylation events to selectively regulate transcription.


Subject(s)
Herpes Simplex Virus Protein Vmw65/chemistry , Transcription Factor TFIIH/chemistry , Tumor Suppressor Protein p53/chemistry , Herpes Simplex Virus Protein Vmw65/genetics , Humans , Mutation , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Structure, Secondary , Protein Structure, Tertiary , Transcription Factor TFIIH/genetics
15.
Nat Struct Mol Biol ; 24(10): 848-856, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28825733

ABSTRACT

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P' helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.


Subject(s)
Enzyme Inhibitors/metabolism , PCSK9 Inhibitors , Peptides/metabolism , Proprotein Convertase 9/metabolism , Binding Sites , Enzyme Inhibitors/isolation & purification , Humans , Molecular Docking Simulation , Peptide Library , Peptides/isolation & purification
16.
J Med Chem ; 60(24): 10056-10070, 2017 12 28.
Article in English | MEDLINE | ID: mdl-29166018

ABSTRACT

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Aminopyridines/chemistry , Binding Sites , Catalytic Domain , Cell Line , Computer Simulation , Crystallography, X-Ray , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , Humans , Magnetic Resonance Spectroscopy/methods , Oxadiazoles/chemistry , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/metabolism
17.
Structure ; 24(4): 498-499, 2016 Apr 05.
Article in English | MEDLINE | ID: mdl-27050685

ABSTRACT

In this issue of Structure, Castañeda et al. (2016b) use multi-disciplinary approaches including NMR techniques, small-angle neutron scattering, and docking to convincingly demonstrate that K27-linked diubiquitin is relatively rigid with unexpected similarity to the conformation of K48-linked diubiquitin bound to the UBA2 domain of hHR23a.


Subject(s)
Models, Molecular , Ubiquitin/chemistry , Magnetic Resonance Spectroscopy , Scattering, Small Angle , Ubiquitins/chemistry
18.
Biomol NMR Assign ; 10(2): 345-9, 2016 10.
Article in English | MEDLINE | ID: mdl-27386854

ABSTRACT

The deubiquitinase Ubiquitin Specific Protease 7 (USP7) is part of the regulatory cascade of proteins that modulates the activity of the tumor suppressor protein p53. Deubiquitination of its target Murine Double Minute 2 (MDM2) leads to increased proteosomal degradation of p53. Consequently, USP7 has emerged as an attractive oncology target because its inhibition stabilizes p53, thereby promoting p53-dependent apoptosis in cancer cells. Here we report the backbone resonance assignment for the 40.5 kDa catalytic domain of USP7.


Subject(s)
Catalytic Domain , Nuclear Magnetic Resonance, Biomolecular , Ubiquitin Thiolesterase/chemistry , Amino Acid Sequence , Humans , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Peptidase 7
19.
Structure ; 24(8): 1335-1345, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27452404

ABSTRACT

The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs.


Subject(s)
Ubiquitin Thiolesterase/chemistry , Ubiquitin/chemistry , Amino Acid Sequence , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Kinetics , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Thermodynamics , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Peptidase 7
20.
ChemMedChem ; 8(5): 726-32, 2013 May.
Article in English | MEDLINE | ID: mdl-23436619
SELECTION OF CITATIONS
SEARCH DETAIL