NMR-Based Discovery of Lead Inhibitors That Block DNA Binding of the Human Papillomavirus E2 Protein

Hajduk, Philip J.; Dinges, Jürgen; Miknis, Gregory F.; Merlock, Megan; Middleton, Tim; Kempf, Dale J.; Egan, David A.; Walter, Karl A.; Robins, Terry; Shuker, Suzy B.; Holzman, Thomas F.; Fesik, Stephen W.

doi:10.1021/jm9703404

Cited by 159 publications

(102 citation statements)

References 23 publications

(54 reference statements)

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“…Resonance assignments obtained at 318 K were transferred to spectra of a longer talin USH-I/LWEQ construct (2300 -2501) at 305 K using standard 3D triple-resonance experiments (40). NMR chemical shift perturbations in spectra recorded at different pH values were classified according to chemical shift changes as medium (orange, ⌬␦TOT ϭ 0.05-0.1 ppm) and large (red, ⌬␦TOT Ͼ 0.1 ppm), were weighted according to ⌬␦TOT ϭ ͉⌬ 1 H͉ ϩ ͉⌬ 15 N͉ ϫ 0.2 (41).…”

Section: Methodsmentioning

confidence: 99%

Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

Srivastava

Barreiro

Groscurth

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

115

121

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Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pHi) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin-actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pKa values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pHi decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin. intracellular pH ͉ NHE1 ͉ migration F ocal adhesion (FA) remodeling is a rate-limiting determinant in haptokinetic migration of adherent cells. At the leading edge of migrating cells, FAs undergo rapid cycles of assembly and turnover, creating and disrupting, respectively, sites of traction necessary for forward movement of the cell body. Force generation for traction requires linkage among the extracellular matrix, integrin receptors, and actin filaments. Actin filaments do not directly bind to the cytoplasmic domain of integrins but bind to integrin-associated FA proteins such as talin and vinculin. Although several mechanisms contribute to FA remodeling in migrating cells (1), emerging evidence indicates that talin plays a central role in the dynamic linkage between integrins and actin filaments necessary for cell migration (2, 3). Talin functions in distinct albeit complementary mechanisms that promote FA turnover. First is cleavage of talin by the protease calpain, which also modulates adhesion complex composition and likely signaling functions of talin (4). Second is regulated talin binding to actin filaments, which is proposed to act as a clutch to control FA turnover and membrane protrusion dynamics (3, 5-7). How actin binding by talin is dynamically regulated during cell migration, however, remains undetermined.Previous stu...

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Section: Methodsmentioning

confidence: 99%

Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

Srivastava

Barreiro

Groscurth

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

115

121

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“…5). The corrected changes in 1 H-15 N chemical shift, upon peptide binding, were calculated as described (23), and shifts of 0.04 ppm and above were considered significant. Changes were observed for Lys-101, Thr-102, and Tyr-103 (from the N terminus); Leu-114, His-115, Gly-117, and Thr-118 (from loop L1); Tyr-126 (from strand S2); Val-143 (from strand S3); Glu-198 and Gly-199 (from strand S5); Arg-248 (from loop L3); Asn-263 and Leu-265 (from strand S10); Gly-279, Arg-280, Arg-282, Arg-283, and Glu-286 (from helix H2); and Lys-291, Lys-292, Gly-293, and Gly-298 (from the C terminus).…”

Section: Defining the Sites Of The Interaction Between P53c And Hdm2-mentioning

confidence: 99%

The central region of HDM2 provides a second binding site for p53

Rüdiger

Veprintsev

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

132

141

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HDM2 is a negative regulator of p53 that inhibits its transcriptional activity and subjects it to degradation by an E3 ligase activity. The primary binding site for HDM2 on p53 is located in its N-terminal domain. A second site on the p53 core domain (p53C) binds to an unidentified site in HDM2. We found that this site is in its acidic domain and part of the zinc finger domain by examining the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. NMR spectroscopy showed that peptides derived from this region of HDM2 bound to residues in the specific DNA-binding site of p53C. The peptides were displaced from the site by gadd45 sequencespecific DNA. Phosphorylation of single amino acids in the central domain of HDM2 did not abolish the interaction between the HDM2-derived peptides and p53C. We speculate that this second binding site helps in stabilizing the interaction between HDM2 and p53 during p53 degradation.isothermal titration calorimetry ͉ Mdm2 ͉ NMR T he tumor suppressor protein p53 is important in maintaining genome stability and in preventing cancer development (1, 2). In response to various stress signals, p53 mediates cell-cycle arrest and apoptosis (3). p53 is a homotetramer consisting of an N-terminal transactivation domain, proline-rich regulatory domain, DNA-binding core domain (p53C), tetramerization domain, and C-terminal negative regulatory domain. Its major regulator, HDM2, is induced by p53 and acts as a feedback inhibitor (4). HDM2 regulates the activity of p53 in at least three ways. First, the N-terminal domain of HDM2 binds directly to p53's transactivation domain and inhibits its transcriptional function (5). Second, HDM2 acts as a ubiquitin ligase, targeting p53 and promoting its degradation (6, 7). Third, upon binding, HDM2 exports p53 from the nucleus to the cytoplasm (8).The interaction between peptides derived from the p53 N terminus and the HDM2-N-terminal domain has been extensively studied (9-12), and several compounds have been proposed to abolish this interaction (13-16). Recently, a new HDM2-binding site has been reported in p53C that plays a regulatory role in modulating p53 ubiquitination (17), although the exact binding site on HDM2 involved in this interaction has not yet been identified. There are conflicting speculations on its location at either the HDM2 N terminus (18) or in the acidic domain (19).Here, we examined the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. We found that the HDM2 N-terminal domain interacts only with p53's N-terminal domain, whereas from the peptide-array screen, we identified a second binding site for p53C located in the central domain (residues 221-302) of HDM2. We measured the binding affinity of the peptides to p53C by analytical ultracentrifugation. We identified the binding site on p53C by NMR and fluorescence anisotropy to be its DNA-binding site....

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“…The purified protein will then be concentrated into PBS buffer, pH 7.4 by centrifugation using either a Centricon YM-3 or Ultra-4 concentrators (Amicon) (Tyler et al, 2005). The availability of 15 N-labeled ERα-LBD protein will allow us to carry out Structure Activity Relationship by NMR (SAR-by-NMR) studies (Hajduk et al, 1997) that will clearly define the binding site and orientation of not only PhIP, but other CBIs and HAs. …”

Section: Tif2 Helixmentioning

confidence: 99%

Computational Characterization and Prediction of Estrogen Receptor Coactivator Binding Site Inhibitors

Bennion¹,

Kulp²,

Cosman³

et al. 2005

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. (From -To) Aug 1 REPORT DATE (DD-MM-YYYY) 01-09-2005 REPORT TYPE Final Report DATES COVERED PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBE RRegents of the University of California Lawrence Livermore Natl. Lab.7000 East Ave Livermore California 94550 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel CommandFort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STAT EMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTES 314. ABSTRACT Many carcinogens have been shown to cause tissue specific tumors in animal models. The mechanism for this specificity has not been fully elucidated and is usually attributed to differences in organ metabolism. For heterocyclic amines, potent carcinogens that are formed in well-done meat, the ability to either bind to the estrogen receptor and activate or inhibit an estrogenic response will have a major impact on carcinogenicity. Here we describe our work with the human estrogen receptor alpha (hERa) and the mutagenic/carcinogenic heterocyclic amines PhIP, MeIQx, IFP, and the hydroxylated metabolite of PhIP, N2-hydroxy-PhIP. We found that PhIP, in contrast to the other heterocyclic amines, increased cell-proliferation in MCF-7 human breast cancer cells and activated the hERa receptor. We show mechanistic data supporting this activation both computationally by homology modeling and docking, and by NMR confirmation that PhIP binds with the ligand binding domain (LBD). This binding competes with estradiol (E2) in the native E2 binding cavity of the receptor. We also find that other heterocyclic amines and N2-hydroxy-PhIP inhibit ER activation presumably by binding into another cavity on the LBD. Moreover, molecular dynamics simulations of inhibitory heterocyclic amines reveal a disruption of the surface of the receptor protein involved with protein-protein signaling. We therefore propose that the mechanism for the tissue specific carcinogenicity seen in the rat breast tumors and the presumptive human brea...

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NMR-Based Discovery of Lead Inhibitors That Block DNA Binding of the Human Papillomavirus E2 Protein

Cited by 159 publications

References 23 publications

Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

The central region of HDM2 provides a second binding site for p53

Computational Characterization and Prediction of Estrogen Receptor Coactivator Binding Site Inhibitors

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