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Molecular and Biochemical Characterisation of Siap As a Sialic Acid Binding Protein Component of a TRAP Transporter for Sialic Acid

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Molecular and Biochemical Characterisation of Siap As a Sialic Acid Binding Protein Component of a TRAP Transporter for Sialic Acid Molecular and biochemical characterisation of SiaP as a sialic acid binding protein component of a TRAP transporter for sialic acid Adam Paul Hopkins A thesis submitted for the degree of Doctor of Philosophy University of York Department of Biology July 2010 Abstract Sialic acid utilisation plays an important role in the growth and persistence of the obligate human mucosal pathogen Haemophilus influenzae, which causes respiratory tract infections, septicaemia and meningitis. Like many other bacteria, H. influenzae can use host-derived sialic acids as carbon, nitrogen and energy sources, but also as a terminal modification on the LPS to better evade the human immune system. H. influenzae takes up exogenous sialic acid via a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM. This possesses an extracytoplasmic substrate binding protein (SBP), SiaP, which binds the substrate in the periplasm and delivers it to the specific membrane permease, SiaQM. SiaP contains two globular domains, which close around the substrate upon binding. Here, the mechanism of sialic acid binding by SiaP is investigated using site-directed mutagenesis of residues in the ligand binding site and analogues of sialic acid. These, and several mutations on the surface of SiaP, were investigated for their effect on transport by SiaPQM in vitro, using SiaQM reconstituted into proteoliposomes, and in vivo, using expression of siaPQM in an E. coli strain lacking its native sialic acid transporter, NanT. It is demonstrated that stabilisation of the carboxylate group of sialic acid by the totally conserved Arginine-147 is important for high-affinity ligand binding, but is not essential for transport. Mutation of Aparagine-150 to Aspartate abolishes the function of the transporter without affecting ligand binding, suggesting the existence of a critical interaction between the components of the transporter. The catabolism of the sialic acid analogues was also examined in E. coli expressing different sialic acid transporters. This indicates that a wide variety of sialic acid analogues are potential carbon sources in many pathogenic bacteria. 2 List of Contents Title page 1 Abstract 2 List of contents 3 List of figures 10 List of tables 13 Acknowledgements 14 Chapter 1. Introduction 15 1.1 The requirement for transporters 16 1.2 Active transport 19 1.3 ATP binding cassette (ABC) transporters 20 1.3.1 The E. coli ABC transporter for maltose as a model system 21 1.3.1.1 The substrate-binding protein (SBP) 21 1.3.1.2 The membrane-associated complex 23 1.3.1.3 The proposed mechanism of transport 24 1.3.2 The role of the PBP in transport 28 1.3.3 The mechanism of PBP domain closure 29 1.3.4 Exploiting SBPs from ABC transporters 32 1.3.4.1 Technological exploitation 33 1.3.4.2 Alternative uses of the PBP fold 35 1.4 Secondary active transport 37 1.4.1 The Major Facilitator Superfamily 37 1.4.2 The E. coli lactose permease as a model system 38 1.4.2.1 The structure of the lactose permease 38 1.4.2.2 Proposed mechanism of transport 40 1.4.3 Sodium-driven co-transport 40 1.5 SBP-dependent secondary active transport 43 1.5.1 Features and components of SBP-dependent secondary transporters 43 1.5.1.1 Co-ordination of the ligand by SBPs 46 3 1.5.1.2 Multimeric SBPs 48 1.5.2 The proposed mechanism of transport 50 1.5.2.1 The R. capsulatus C4-dicarboxylate transporter, DctPQM 50 1.5.2.2 The H. influenzae sialic acid transporter, SiaPQM 50 1.6 Sialic acids 52 1.6.1 Utilisation and catabolism of sialic acids 54 1.6.1.2 Use of sialic acid by H. influenzae 56 1.7 Aims of this investigation 57 Chapter 2. Materials and Methods 59 2.1 Media and antibiotics 60 2.1.1 Luria-Bertani broth 60 2.1.2 M9 minimal medium 60 2.1.3 Solid media 60 2.1.4 Antibiotics 60 2.2 Table of primers 60 2.3 Strains and plasmids 60 2.4 General cloning techniques 66 2.4.1 Agarose gel electrophoresis 66 2.4.2 Plasmid preparation 66 2.4.3 Polymerase Chain Reaction (PCR) 66 2.4.4 Site-directed mutagenesis PCR 66 2.4.4.1 Mutagenic primer design 66 2.4.4.2 Mutagenic PCR conditions 67 2.4.5 Megaprimed mutagenic PCR 67 2.4.5.1 Megaprimer primer design 67 2.4.5.2 Megaprimer PCR 67 2.4.5.3 Megaprimed mutagenic PCR 67 2.4.6 Preparation of DNA fragments and cloning 68 2.4.6.1 Restriction enzyme digestions 68 2.4.6.2 Dephosphorylation reaction conditions 68 4 2.4.6.3 Gel extraction 68 2.4.6.4 PCR clean up 68 2.4.6.5 Ligation of DNA 68 2.4.7 Transformations 69 2.4.7.1 Chemically competent E. coli DH5α stock 69 2.4.7.2 Small volumes of chemically competent cells 69 2.4.7.3 Heat shock 69 2.4.7.4 Freeze-thaw competency 69 2.5 Growth of bacteria 70 2.5.1 Expression of periplasmic proteins from pET-based constructs 70 2.5.2 Expression of cytoplasmic proteins from pRSET-based constructs 70 2.5.3 Expression of membrane proteins from pBAD-based constructs 70 2.6 Preparation of bacterial extracts 71 2.6.1 Periplasmic fraction preparation 71 2.6.2 Cytoplasmic protein recovery 71 2.6.3 Preparation of cell membranes as total membrane vesicles 71 2.7 Protein purification techniques 71 2.7.1 Hydrophobic Interaction Chromatography (HIC) 71 2.7.2 Size Exclusion Chromatography (SEC) 72 2.7.3 Nickel-affinity chromatography 72 2.7.3.1 FPLC 72 2.7.3.2 Peristaltic pump 73 2.7.4 Nickel-Nitrilotriacetic acid (Ni-NTA) resin – FLIP 73 2.7.4.2 Purification of soluble protein 73 2.7.4.2 Purification of membrane protein 74 2.7.5 Reconstitution of membrane protein into proteoliposomes 74 2.8 Large volume fermentation for the production of SiaP-His6:A11N 74 2.8.1 Expression of protein from the pAH55 construct 74 2.8.2 Preparation of bacterial extracts 75 2.9 Sodium docecylsulphate polyacrylamide gel electrophoresis (SDS 75 PAGE) 5 2.10.1 Native conditions 75 2.9.1.1 Buffers and gel 75 2.9.1.2 Sample preparation 75 2.9.1.3 Running conditions 75 2.9.2 Denaturing conditions 75 2.9.2.1 Buffers and gel 76 2.9.2.2 Sample preparation 76 2.9.2.3 Running conditions 76 2.9.3 Staining/destaining 76 2.9.4 Western blotting 76 2.9.4.1 Buffers 76 2.9.4.2 Running conditions 77 2.9.4.3 Sample visualisation 77 2.10 Fluorescence spectroscopy 77 2.11 Isothermal Titration Calorimetry (ITC) 78 2.11.1 ITC protocol 78 2.11.2 ITC for SiaP-His6:F170W 78 2.11.3 ITC for higher Kd values 79 2.12 Circular Dichroism (CD) 79 2.12.1 CD spectra determination 79 2.12.2 Thermal stability by CD 79 2.13 In vivo growth assays 79 2.13.1 Growth on solid medium 79 2.13.2 Liquid culture 79 2.13.3 Prototype incubated plate shaker – buffers etc and pre-growth 80 2.13.3.1 24-well plate set up 80 2.13.3.2 The prototype incubated plate shaker 80 2.13.3.3 Standard curve 80 2.14 ELISA-based assays for the detection of SiaP-His6 80 2.14.1 Target substrates 80 2.14.2 96-well plate assay 81 6 2.15 14C-radiolabelled sialic acid-based assays 81 2.15.1 Filter binding assay 81 2.15.2 In vitro 14C-sialic acid transport assay 81 2.15.2.1 Preparation of SiaQM-containing proteoliposomes 81 2.15.2.2 Buffers for gradients 83 2.15.2.3 Standard assay 83 2.15.2.4 Altered 14C-sialic acid concentration 83 2.15.2.5 Competition assay 83 2.16 Guanidine hydrochloride (GnHCl) denaturation for protein recycling 84 2.16.1 Buffers 84 2.16.2 In-column denaturation and refolding 84 Chapter 3. Examination of the contribution of individual amino acid residues 85 to high-affinity sialic acid binding by SiaP 3.1 Strategy for site-directed mutagenesis of the siaP gene to alter sialic 86 acid-binding properties of SiaP 3.2 Introduction of Arginine-147 mutants into native SiaP 86 3.3 SiaP-His6 functions as SiaP both in vitro and in vivo 91 3.4 The introduction of tryptophan residues can improve the 96 fluorescence signal change on ligand binding 3.5 Mutations of residues co-ordinating the ligand carboxylate group 100 disrupt ligand binding 3.6 An aromatic residue is required at position 170 for high-affinity ligand 104 binding 3.7 Co-ordination of the ligand carboxylate group is important for 106 high-affinity binding 3.8 No ligand binding can be detected for the Arg-147 mutants in vitro 109 3.9 The Arg-147 residue is not essential for sialic acid transport in vivo 115 3.10 Summary 118 7 Chapter 4. Investigation and manipulation of the ligand binding properties 120 of SiaP for application as a biosensor 4.1 Investigation of the biophysical properties of SiaP-His6 121 4.1.1 Ethanol decreases ligand binding affinity and promotes α-helix formation 122 4.1.2 Ligand binding affinity is affected by ionic strength and the presence of 125 sodium ions 4.1.3 Decreasing temperature increases ligand binding affinity 129 4.1.4 Sialic acid-binding by SiaP is enthalpically driven and releases water 131 4.1.5 Optimised conditions for high affinity binding of Neu5Ac by SiaP 134 4.2 Detection of sialic acid 136 4.2.1 The Lateral Flow Device 136 4.2.2 Conjugated sialic acid as a basis for a Lateral Flow Device 136 4.2.3 SiaP is specific for monomeric sialic acid 138 4.2.4 An in-solution approach for sialic acid detection 143 4.3 Rational design of SiaP to modulate its sialic acid binding affinity 145 4.3.1 Binding site mutations designed to increase ligand affinity 145 4.3.2 Mutations designed to promote the closed conformation 147 4.4 Summary 151 Chapter 5.
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