Structural biology of Cystic Fibrosis Transmembrane Conductance Regulator, an ATP-binding cassette protein of medical importance

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein that functions as an ion channel. Mutations in this protein cause Cystic Fibrosis. For this reason, it is important to study the structure and function of CFTR. In this study, constructs of CFTR (C-terminii), a CFTR-interacting protein and full-length CFTR were cloned, expressed and purified for structural and functional studies. The purified C-terminal polypeptides of CFTR were soluble and shown to interact with NHERF1 PDZ 1 (a CFTR-interacting protein). The CFTR C-terminus and NHERF1 PDZ 1 domain were co-expressed and co-purified. The purified complex showed a strong interaction that might induces a conformational change. Site-directed mutation of the C-terminus of CFTR was performed in order to examine the effect of removing a potentially flexible amino acid (Arginine) on protein crystallization. Pull-down assay experiments with full-length CFTR demonstrated an interaction between CFTR (in DDM detergent) and NHERF1 PDZ 1(+). No interaction was observed for CFTR in LPG (a relatively denaturing detergent) and NHERF1, implying that the interaction between the PDZ motive of CFTR and NHERF1 requires a stable folded structure for both proteins. In addition, full-length CFTR in DDM has been studied by electron microscopy and Single Particle Analysis in the presence of NHERF1 PDZ 1(+). A 3D structure was generated for the CFTR-NHERF1 PDZ 1(+) complex at a resolution of ~ 18 Ã…. This 3D structure showed a new open conformation of CFTR (V shape). In comparable studies with CFTR alone, a 3D structure was generated at a resolution of 27 Ã… and this structure showed a closed state as previously reported. This new data suggest a possible role for NHERF1 in terms of CFTR channel gating or activation.

Layman's Abstract

Table of ContentsAcknowledgements 3Table of Contents 4List of Tables 11List of Figures 12List of Abbreviations 17Introduction 211.1 Transmembrane proteins 211.2 ABC proteins 231.3 The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) 251.3.1 CFTR-Interacting Proteins 291.3.1.1 CFTR-NHERF Interactions 301.3.1.1.1 Does NHERF-C-terminus of CFTR binding have an important role in CF? 341.4 Structural Biology of Transmembrane Proteins 351.4.1 Electron Microscpy 351.4.1.1 2D Crystals of CFTR 371.4.1.2 Single Particle Analysis of CFTR 391.4.2 The Structure of CFTR Domains 421.4.2.1 Prediction of CFTR Topology (and the location of C-terminus) 461.4.2.2 The C-terminus of CFTR 471.4.2.3 Expression and Purification of CFTR 491.5 Characterization of Protein Using Circular Dichroism 501.6 Characterization of Protein by MALDI-TOF Mass Spectrometry 541.7 Aims and Objectives of this Study 56Materials and Methods 582.1 Materials 582.1.1 Source of the CFTR Protein 582.1.2 Growth Media 582.1.3 Antibiotics 582.1.4 Inducers 592.1.5 Buffers and Solutions 592.1.5.1 Buffer A (Unbound Materials Washing Buffer) 592.1.5.2 Buffer B (Elution Buffer) 592.1.5.3 Buffer C (Sonication Buffer) 592.1.5.4 Dialysis Buffer 592.1.5.5 GST (the glutathione S-transferase) tag Purification Buffers 592.1.5.6 Gel Filtration Buffer 592.1.5.7 CFTR Buffer (DDM) 592.1.5.8 CFTR Buffer (LPG) 602.1.5.9 Phosphate Buffer 602.1.5.10 Thrombin Buffer 602.1.5.11 TFB1 Buffer 602.1.5.12 TFB2 Buffer 602.1.5.13 Uranyl Acetate Solution 602.1.5.14 Super Blue Coomassie Stain 602.1.5.15 15 % Tricine Gel Buffers 602.1.5.16 Agarose Gel Buffers 612.1.5.17 M9 / Minimal Media for NMR (Nuclear Magnetic Resonance) Analysis 612.1.6 E. coli Strains and Vectors 622.1.7 PCR Primers (Oligonucleotides) 632.2 Methods 642.2.1 Designing Primers 642.2.1.1 P1For and P2Rev Primers 642.2.1.2 R-AFor and R-ARev Primers 642.2.1.3 Shark and Killifish Forward and Shark Reverse Primers 642.2.2 Recombinant DNA Processing and Manipulation 652.2.2.1 PCR Amplification of DNA Segments of Interest 652.2.2.2 Insert Restriction Digestion 652.2.2.3 Vector Restriction Digestion 662.2.2.4 Ligation and Transformation 662.2.2.5 Site-Directed Mutagenesis (Quick Change) 672.2.2.6 Gel Electrophoresis 672.2.3 Preparation of Competent E. coli 682.2.4 Expression and Purification 682.2.4.1 Small-Scale Expression of Targets 682.2.4.2 Large-Scale Expression of Targets 692.2.4.3 Protein Purification Using Metal Affinity Chromatography 692.2.5 Protein Concentration 702.2.6 SDS-PAGE Analysis 712.2.7 MALDI-TOF-MS Analysis 712.2.8 Circular Dichroism Analysis 722.2.9 NMR Spectroscopy Analysis 722.2.10 Histidine-tag Pull-down Assay 732.2.11 Gel Filtration of NHERF1 PDZ 1 732.2.12 Vapour Diffusion Method (sitting drop) 742.2.13 Vapour Diffusion Method (hanging drop) 742.2.14 Single Particle Analysis Electron Microscopy 752.2.14.1 Glow Discharging and Negative Staining of Samples 752.2.14.2 Electron Microscopy 762.2.14.3 Single Particle Analysis and 3D Reconstruction 762.2.14.4 Particle Selection 762.2.14.5 Classification of Particles 772.2.14.6 Reconstruction of a Preliminary 3D Model 782.2.14.7 Refinement of the 3D Model 782.2.14.8 Processing commands used in SPA 792.2.15 CFTR Reconstitution by Freeze-Thaw 82Results and Discussion 833.1 Results and Discussion 1 833.1.1 Prediction of Secondary Structure of the C-terminus42aa 833.1.2 Expression, Purification, Characterization and Crystallization Trials of the C-terminus42aa 843.1.2.1 Expression and Purification of the C-terminus42aa 843.1.2.2 Characterization of the C-terminus42aa 863.1.2.2.1 MALDI-TOF-MS 863.1.2.2.2 Circular Dichroism measurement of the C-terminus42aa 913.1.2.2.3 NMR Data 913.1.3 Co-expression, Co-purification, and Co-characterization of the C-terminus42aa of Human CFTR and NHERF1 PDZ1 933.1.3.1 Sub-cloning of NHERF1 PDZ 1 into pET-52b 933.1.3.2 Co-expression and Co-purification of the C-terminus42aa and NHERF1 PDZ 1 933.1.3.3 Characterization of the C-terminus42aa and NHERF1 PDZ 1 963.1.3.3.1 A dimerzation of the C-terminus42aa and NHERF1 PDZ 1 963.1.3.3.2 MALDI-TOF-MS 973.1.3.3.3 Circular Dichroism of the C-terminus42aa and C42aa-NHERF1 PDZ 1 complex 1003.1.3.3.4 Does the C-terminus42aa-NHERF1 PDZ 1 interaction require more contact than QDTRL-NHERF1 PDZ 1 interaction? 1023.1.4 Study of Constructs in terms of Expression and Purification 1063.1.4.1 Expression and Purification of the C-terminus61aa 1063.1.4.1.1 Expression and Purification of the C-terminus61aa 1073.1.4.2 Expression and Purification of the C-terminusR-A 1103.1.4.2.1 Determination of Flexibility of Surface Amino Acids 1103.1.4.2.2 Site-directed Mutagenesis of the C-terminusR-A 1123.1.4.2.3 Expression and Purification of the C-terminusR-A 1143.1.4.3 Sub-cloning, Expression and Purification of Shark C-terminus 1153.1.4.3.1 Sub-cloning of Shark C-terminus into pGA4 Vector 1163.1.4.3.2 Expression and Purification of Shark C-terminus 1173.1.4.4 Sub-cloning, Expression and Purification of Killifish C-terminus 1173.1.5 Expression and Purification of NHERF1 PDZ 1 1193.1.5.1 Gel Filtration Chromatography of NHERF1 PDZ 1 1213.2 Results and Discussion 2 1243.2.1 Pull-down Assay of CFTR-NHERF1 PDZ 1 1243.2.1.1 CFTR-NHERF1 PDZ 1 Interaction Analysis 1253.2.1.1.1 Pull-down Assay for Human CFTR-NHERF1 PDZ 1 Complex 1253.2.1.1.2 Pull-down Assay for Mouse and Killifish CFTR-NHERF1 PDZ 1 Complex 1283.2.1.1.3 Pull-down Assay for Human CFTR-C-terminus42aa Complex 1313.2.1.1.4 Pull-down Assay for killifish CFTR-C-terminus42aa Complex 1333.2.2 Single Particle Analysis of Human CFTR-NHERF1 PDZ 1 1373.2.2.1 A Low Resolution Structure of CFTR-NHERF1 1373.2.2.1.1 Analysis of the 3D Structure of CFTR-NHERF1 PDZ 1 1453.2.2.2 A Low Resolution Structure of Isolated CFTR 1503.2.2.3 Structural Analysis of the CFTR-NHERF1 and the Isolated CFTR Structures 1593.2.2.3.1 Distance Measurement 1593.2.2.3.2 Structural Similarity 1613.2.2.3.3 The C-terminus Location 1633.2.2.3.4 CFTR channel gating 1643.2.2.3.5 Dimerization of CFTR 164Conclusion 166Future Work 172Appendix A 174Appendix B 179Appendix C 181pGEX-2T vector 181pET-52b vector map 183pET-24a vector map 184pGA4 vector map 185Appendix D 186Sitting drop experiments 186Hanging drop experiments 186Orbi-trap Mass Spectrometry of the C-terminus42aa-NHERF1 PDZ 1 189Analysis of CD data by DichroWeb 190Micrographs of the reconstituted mouse and killifish CFTR 195References 197

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Protein expression and Purification, Electron Microscopy, Single Particle Analysis.

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