Structural Investigations of Proteins Involved in the COPI Complex
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Structural investigations of proteins involved in the COPI complex Avital Lahav Structural investigations of proteins involved in the COPI complex Research Thesis Submitted In Partial Fulfillment of The Requirements for the Degree of Doctor of Philosophy Avital Lahav Submitted to the Senate of the Technion – Israel Institute of Technology Kislev, 5774 Haifa November 2014 The research was done under the supervision of Prof. Noam Adir in the Schulich Faculty of Chemistry. First and foremost I would like to thank my supervisor Prof. Noam Adir for his help, guidance and understanding during all my research period. Second, I would like to thank Prof. Dan Cassel from the Biology Faculty in the Technion for his collaboration and help on this research and to Dr. Anna Parnis from Prof. Dan Cassel's lab for her help and patience during our work. Third, I would like to thank my friends from the lab of both Prof. Noam Adir and Prof. Dan Cassel who made the lab a pleasant place to work at and from I learned a lot. The generous financial help of the Technion is gratefully acknowledged. I would like to thank my wonderful and beloved family: Yehuda, Ben and Adam who with one hug make everything right. This thesis is dedicated to them. To David Klein who again, helps me reach the stars. CONTENTS Abstract 1 Abbreviations 3 1. Introduction 5 1.1 The COPI system of vesicular traffic 5 1.1.1 The GTPase-activating proteins, ArfGAPs 6 1.1.2 The γCad 8 1.1.3 The MHD of the δ–COPI subunit 9 1.1.4 The R-based signals recognition site 10 1.1.5 The ArfGAP1 interaction with δCad 11 1.2 Single crystal X-ray crystallography 12 1.2.1 Protein's preparation 12 1.2.2 Crystal mounting 13 1.2.3 X-ray source 14 1.2.4 Basic concepts 14 1.2.4.1 The unit cell 14 1.2.4.2 The asymmetric unit 14 1.2.4.3 The space group 15 1.2.4.4 The reciprocal lattice 15 1.2.4.5 Bragg's law and Miller indices 15 1.2.5 Data processing 16 1.2.6 Scaling 17 1.2.7 Solutions to the phase problem 18 1.2.8 Calculation of the electron density map 18 1.2.9 Model building 19 1.2.10 Refinement 19 1.2.11 Model validation 20 2. Research goals 21 3. Research plan and methods 22 3.1 Research plan 22 3.2 Methods 23 3.2.1 Molecular methods 23 3.2.1.1 Preparation of competent cells 23 3.2.1.2 PCR- Polymerase Chain Reaction 23 3.2.1.3 Agarose gel electrophoresis of DNA 23 3.2.1.4 DNA digestion by restriction enzymes 24 3.2.1.5 Ligation of PCR product into cloning vector 24 3.2.1.6 Electro-transformation 24 3.2.1.7 DNA sequence determination 24 3.2.2 Protein Isolation 24 3.2.2.1 Overexpression of the proteins 24 3.2.2.2 Isolation and primary purification of the proteins 25 3.2.2.3 SDS-PAGE analysis 25 3.2.2.4 Chromatographic analysis 25 3.2.2.5 Protein concentration 26 3.2.2.6 Determination of protein concentration 26 3.2.2.7 Mass spectroscopy 26 3.2.3 Protein analysis 26 3.2.3.1 SLS measurements 26 3.2.3.2 ITC measurements 27 3.2.4 Crystallization of the proteins 27 3.2.4.1 Improving crystal growth 27 3.2.4.2 Twinning and implementing twin law 28 3.2.5 Software used in crystallographic structure determination 28 3.2.5.1 Data processing, scaling and refinement 28 3.2.5.1.1 Mosflm 28 3.2.5.1.2 XDS 28 3.2.5.1.3 HKL-2000 package 28 3.2.5.1.4 CCP4 29 3.2.5.1.5 CNS 29 3.2.5.1.6 Phenix 29 3.2.5.2 Molecular graphic tools 29 3.2.5.2.1 PyMol 29 3.2.5.2.2 Coot 29 3.2.5.3 Bioinformatics programs 30 3.2.5.3.1 BLAST 30 3.2.5.3.2 Generunner 30 3.2.5.3.3 Clone Manager 30 3.2.5.3.4 WebCutter 30 3.2.5.3.5 PHYRE2 30 3.2.5.3.6 SWISS-MODEL 30 3.2.5.3.7 PDBePISA 30 4. Materials 31 4.1 General reagents 31 4.2 Vitamins 32 4.3 Amino acids 32 4.4 Biochemical and crystallization kits 33 4.5 Enzymes 33 4.6 Other materials 33 4.7 Growth media 34 4.8 Cell lines 34 4.9 Plasmid vectors and oligonucleotides 35 4.9.1 Vector description 35 4.9.2 Oligonucleotides 35 4.10 Buffers and solutions 37 4.10.1 Solutions for electrophoresis of DNA on agarose gel 37 4.10.2 Buffers used for protein isolation, purification and biochemical experiments 37 4.10.2.1 Nickel column buffers 37 4.10.2.2 Gel filtration buffers 38 4.10.3 Buffers used for SDS-PAGE 38 4.11 Gel recipes 38 4.12 Column material and sources 38 5. Results 39 5.1 The interaction between γCad and ArfGAP2 39 5.1.1 γCad and ArfGAP2 co-crystallization 39 5.1.1.1 γCad 39 5.1.1.2 ArfGAP2 39 5.1.1.3 Co-crystallization trials 40 5.1.2 The γCad and ArfGAP2 fusion protein 46 5.1.2.1 Designing the fusion protein 46 5.1.2.2 The fusion protein interaction experiments. 47 5.1.2.3 The fusion protein crystallization 50 5.1.2.4 Analysis of the putative ArfGAP2- γCad interaction site 54 5.2 The δCM 56 5.2.1 δCM purification 56 5.2.2 δCM crystallization 57 5.2.3 δCM data determination 59 6. Discussion 67 6.1 The interaction between γCad and ArfGAP2 67 6.1.1 Adding unstructured protein for crystallization 67 6.1.2 Findings of interaction. 67 6.1.3 Suggestions for the future. 68 6.2 The δCM 68 6.2.1 The δCM structure for interpretation of the entire COPI system 68 6.2.2 Exploration of the δCM interface and assembly 72 6.2. Mutation in δCM involved in Neurodegenerative disorder 72 7. References 74 LIST OF FIGURES Figure Title Page 1 Model for COPI Assembly 6 2 Identification of residues in the basic stretch of ArfGAP2/3 that are important 7 for ER localization 3 Potential protein interaction sites on γCad 9 4 A composite model of F-COPI subcomplex 10 5 Growing protein crystals by vapor diffusion 13 6 Crystal organization 15 7 Conditions that produce strong diffracted rays 16 8 SDS_PAGE gel of γCad purification 39 9 Crystal lattice packing of the two γCad crystal structures 41 10 Structural comparison between γCad structure (1R4X) and the structure 46 determined from the co-crystallization 11 Cloning information of the different lengths of the fusion protein 47 12 ArfGAP2 competition with γCad, Fusion protein and ArfGAP2-II peptide. 48 13 Calorimetric data for the binding of ArfGAP2-II peptide to the γCad and the 49 "Medium" length Fusion protein 14 Crystals containing the “Medium” length fusion protein 50 15 Crystal structure of γCad from "Medium" fusion protein overlaid by 2Fo-Fc 53 and Fo-Fc electron density maps showing unoccupied density 16 γCad and ArfGAP2 structure electrostatics 55 17 Structural comparison between the γCad structure (1R4X) and the structure 56 determined from “Medium” length Fusion protein. 18 δCM purification 57 19 δCM crystal MS analysis 58 20 δCM diffraction pattern 59 21 Preliminary model fitting of δCM to the electron density map 60 22 Model building of δCM 62 23 Crystal structure of δCM protein 63 24 Section of the δCM protein crystal structure overlaid by 2Fo-Fc electron 65 density map 25 MHD structural differences 66 26 Partial model for open conformation of Coatomer 70 27 Proposed R-based signals binding site visualized in δCM structure 69 28 Modelling of the I422T point mutation in δCM 73 LIST OF TABLES Table Title Page 1 Oligonucleotides details 36 2 Crystals obtained from published and novel crystallization 42 conditions 3 Structures solved from crystals containing γCad and ArfGAP-II 44 peptide 4 Data collection and refinement statistics for fusion protein 51 5 Different crystals obtained from different crystallization conditions 56 6 Se atoms coordinates for SAD phasing 60 7 Data collection and refinement statistics for the δCM 64 LIST OF EQUATIONS Equation Title Page 1 Bragg’s law 16 2 Mattews coefficient 17 3 Electron density as a Fourier series 19 4 R-factor 19 ABSTRACT The complex set of coordinated and regulated chemical reactions present in all living cells, is called metabolism. As the environments of most organisms are constantly changing, the primary role of these processes is to provide the necessary energy to fuel and maintain the organism’s functions, allowing organisms to respond to their environments, grow and reproduce. To understand cellular processes one needs to delineate how the responses are transmitted and how molecular factors perform specific roles within the cell, through the hierarchy of assemblies and higher structures. One of the ways to elucidate these properties is by the collection of structural data provided from X-ray crystallography. In this research thesis, we studied two proteins that are part of one system; both may serve as a tool for understanding an important cellular process.