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Structural Analysis of the DAP5 MIF4G Domain and Its Interaction with Eif4a

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Structural Analysis of the DAP5 MIF4G Domain and Its Interaction with Eif4a Structural Analysis of the DAP5 MIF4G Domain and Its Interaction with eIF4A Geneviève Virgili Department of Biochemistry Groupe de Recherche Axé sur la Structure des Protéines (GRASP) McGill University Montreal, QC April 2013 ________________________________________________________________________ A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science under the Faculty of Medicine © GENEVIÈVE VIRGILI, 2013 Never trust an atom. They make up everything. - Unknown author ii List of abbreviations 4E-BP eIF4E binding-protein ATP adenosine triphosphate ATPase adenosine triphosphatase Bcl B cell lymphoma CBP80 cap-binding protein 80 CHESS Cornell high energy synchrotron source Csk casein kinase CDK1 cyclin-dependent kinase 1 DAP5M MIF4G domain of DAP5 DNA deoxyribonucleic acid DTT dithiothreitol eIF eukaryotic initiation factor EMCV encephalomyocarditis virus EMSA electrophoretic mobility shift assay Erk extracellular signal-regulated kinases GTP guanosine triphosphate HCV Hepatitis C virus HEAT Huntingtin, Elongation factor 3, A subunit of protein phosphatase 2A [PP2A], and Target of rapamycin IAP inhibitor of apoptosis protein IPTG isopropyl-β-D-thiogalactoside ITC isothermal titration calorimetry Met methionine (or methionyl) MIF4G middle domain of eIF4G mRNA messenger RNA mRNP mRNA-protein complex Mnk MAPK-interacting kinase mTOR mammalian target of rapamycin NOM1 nucleolar MIF4G domain-containing protein 1 NOT1 negative regulator of transcription subunit 1 iii ORF open reading frame PABP poly(A) binding protein Paip1 PABP-interacting protein 1 PDB Protein Data Bank Pdcd4 programmed cell death protein 4 PI3K phosphatidylinositol 3-OH kinase PIC pre-initiation complex PKC protein kinase C PKR protein kinase double-stranded RNA-dependent PMSF phenylmethylsulfonyl fluoride RENT2 regulator of nonsense transcripts 2, also known as Upf2 RNA ribonucleic acid RRM RNA-recognition motif S6K ribosomal protein S6 kinase SAXS small-angle X-ray scattering SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Ser serine TC ternary complex TEV Tobacco Etch virus Thr threonine tRNA transfer RNA Met tRNAi initiator tRNA UTR untranslated region Val valine iv Abstract Death-associated protein 5 (DAP5/p97) is a homolog of the eukaryotic initiation factor 4G (eIF4G) that promotes the IRES-driven translation of multiple cellular mRNAs. Central to its function is the middle domain (MIF4G), which recruits the RNA helicase eIF4A. The middle domain of eIF4G consists of tandem HEAT repeats that coalesce to form a solenoid-type structure. Here, we report the crystal structure of the DAP5 MIF4G domain. Its overall fold is very similar to that of eIF4G, however, significant conformational variations impart distinct surface properties that could explain the observed differences in IRES binding between the two proteins. Interestingly, quantitative analysis of the DAP5-eIF4A interaction using isothermal titration calorimetry reveals a 10-fold lower affinity than with the eIF4G-eIF4A interaction that appears to affect their ability to stimulate eIF4A RNA unwinding activity in vitro. This difference in stability of the complex may have functional implications in selecting the mode of translation initiation. v Résumé Death-associated protein 5 (DAP5/p97) est une protéine homologue au facteur d'initiation eIF4G qui soutient la traduction d'ARN cellulaires contenant des éléments IRES chez les eukaryotes. Au centre de sa fonction est le domaine MIF4G, qui recrute l'ARN hélicase eIF4A. Ce domaine est constitué de motifs HEAT qui se répètent en tandem pour former une structure en forme de solénoïde. Nous rapportons ici la structure cristalline du domaine MIF4G de DAP5. Sa structure est très similaire à celle d’eIF4G. Toutefois, d'importantes variations de conformation lui confèrent des propriétés de surface distinctes qui pourraient expliquer les différences de liaison d'IRES observées entre les deux protéines. Notamment, l'analyse quantitative de l'interaction DAP5-eIF4A par calorimétrie de titration isotherme révèle une affinité 10 fois plus faible que l'interaction eIF4G-eIF4A, ce qui semble affecter la capacité de DAP5 à stimuler l'activité hélicasique d'eIF4A in vitro. Cette différence dans la stabilité du complexe pourrait être impliquée dans la sélection du mode d'initiation de la traduction. vi Table of contents List of abbreviations..........................................................................................................iii Abstract................................................................................................................................v Résumé ..............................................................................................................................vi Table of contents...............................................................................................................vii List of tables and figures..................................................................................................ix Preface..................................................................................................................................x Acknowledgements.............................................................................................................xi Prologue: Regulation of gene expression from DNA to protein..........................................1 Chapter 1: Basic concepts in the study of translation initiation 1 Translation........................................................................................................................4 1.1 Eukaryotic translation initiation.....................................................................................6 1.1.1 Anatomy of the mRNA...................................................................................6 1.1.2 General mechanism of eukaryotic translation initiation.................................7 1.1.3 Components of eIF4F.....................................................................................9 1.1.3.1 Regulation of eIF4F.........................................................................9 1.1.3.2 eIF4E..............................................................................................10 1.1.3.3 eIF4G.............................................................................................10 1.1.3.4 eIF4A.............................................................................................14 1.2 The versatility of eIF4G family and eIF4G-like proteins............................................18 1.2.1 Occurrence of eIF4G domains in other proteins...........................................18 1.2.2 Role of the MA3 domain of eIF4G...............................................................19 1.2.3 Alternative modes of ribosome recruitment.................................................23 1.2.3.1 Viral infection ...............................................................................23 1.2.3.2 Cap-independent translation of cellular mRNAs...........................24 1.3 Death-Associated-Protein 5 (DAP5)............................................................................27 1.3.1 Topology of DAP5........................................................................................27 1.3.2 Function in cap-independent translation initiation.......................................28 1.3.3 Three-dimensional structure.........................................................................30 vii 1.4 Rationale of the study..................................................................................................32 Chapter 2: Structural analysis of the DAP5 MIF4G domain and its interaction with eIF4A 2.1 Introduction..................................................................................................................34 2.2 Results..........................................................................................................................38 2.2.1 Overall structure of the DAP5 MIF4G domain, or DAP5M........................38 2.2.2 Comparison of DAP5M and eIF4G MIF4G domains...................................42 2.2.3 Identification of a potential IRES binding site in DAP5M...........................43 2.2.4 The sites of interaction with eIF4A are structurally conserved in the middle domains of eIF4G and DAP5 but have different binding affinities.......................47 2.2.5 Mutational analysis of Site 1 and Site 2 residues in the DAP5-eIF4A complex..................................................................................................................50 2.2.6 Effect of DAP5M on the helicase activity....................................................53 2.3 Discussion....................................................................................................................55 2.4 Materials and methods.................................................................................................56 2.4.1 Expression and purification of recombinant proteins...................................56 2.4.2 Mutagenesis of DAP5M...............................................................................58 2.4.3 Crystallization and data collection................................................................58 2.4.4 Structure determination.................................................................................58 2.4.5 Gel filtration chromatography.......................................................................59 2.4.6 In vitro pull-down
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