STRUCTURE AND MECHANISM OF THE BACTERIAL TRANSPORTER LEUT By Chayne L. Piscitelli A DISSERTATION Presented to the Department of Biochemistry and Molecular Biology and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy January 2011 School of Medicine Oregon Health & Science University CERTIFICATE OF APPROVAL This is to certify that the Ph.D. dissertation of Chayne L. Piscitelli has been approved ______________________________________ Eric Gouaux, Mentor/Advisor ______________________________________ David Farrens, member ______________________________________ Buddy Ullman, member ______________________________________ Francis Valiyaveetil, member ______________________________________ Ujwal Shinde, member TABLE OF CONTENTS Acknowledgements ........................................................................................................ v List of Abbreviations ..................................................................................................... vi Abstract ............................................................................................................................ ix Chapter 1 Introduction and Fundamental Concepts .................................................................. 1 Crossing the membrane ............................................................................................. 2 Sodium-coupled secondary transport ....................................................................... 5 Neurotransmitter uptake by sodium-coupled secondary transporters ................... 7 Cloning of neurotransmitter transporters defines the NSS family .......................... 9 Disease and pharmacology of NSS family members ............................................... 11 Transport kinetics and thermodynamic coupling .................................................. 15 The crystal structure of LeuT .................................................................................. 20 Preview of this dissertation ..................................................................................... 24 Figures ......................................................................................................................... 28 Chapter 2 Substrate Specificity and Competitive Inhibition of LeuT .................................... 36 Abstract ....................................................................................................................... 37 Introduction ............................................................................................................... 38 Materials and Methods ............................................................................................. 40 Protein expression and purification for functional experiments ........................... 40 Preparation of LeuT proteoliposomes ..................................................................... 40 Inhibition of binding and transport screens ........................................................... 40 Saturation binding ................................................................................................... 41 i Competition binding ................................................................................................ 41 Steady-state kinetics ................................................................................................ 42 Tryptophan time course ........................................................................................... 42 Binding versus transport time course ..................................................................... 43 Inhibition kinetics .................................................................................................... 43 Preparation of LeuT-amino acid complex crystals ................................................. 43 Structure determination .......................................................................................... 45 Conformational shift analysis .................................................................................. 46 Results and Discussion ............................................................................................. 47 Substrate screen of LeuT .......................................................................................... 47 LeuT-substrate crystal structures reveal an occluded state .................................. 48 LeuT-Trp complex adopts an open-to-out conformation ....................................... 50 Second ligand binding site is unique to the open-to-out conformation ................. 52 A model for transport and inhibition ...................................................................... 53 Tables .......................................................................................................................... 57 Figures ......................................................................................................................... 60 Movie Legend ............................................................................................................. 73 Chapter 3 Stoichiometry of Substrate Binding to LeuT ........................................................... 74 Introduction ............................................................................................................... 75 Materials and Methods ............................................................................................. 76 Mutagenesis and protein purification ..................................................................... 76 Isothermal titration calorimetry ............................................................................. 77 Equilibrium dialysis ................................................................................................. 78 ii Scintillation proximity assays .................................................................................. 78 Transport time course.............................................................................................. 79 Steady-state kinetics ................................................................................................ 80 Results and Discussion ............................................................................................. 81 LeuT:substrate stoichiometry is 1:1 ......................................................................... 81 L400 mutants bind leucine equivalent to wildtype .................................................... 83 Clomipramine does not inhibit leucine binding ........................................................ 84 Transport activity is consistent with single-site kinetics .......................................... 85 Tables .......................................................................................................................... 88 Figures ......................................................................................................................... 96 Chapter 4 Engineering Tryptophan Transport Activity into LeuT ...................................... 102 Abstract ..................................................................................................................... 103 Introduction ............................................................................................................. 104 Materials and Methods ........................................................................................... 106 Homology modeling of TnaT ................................................................................. 106 Expression and purification ................................................................................... 107 Preparation of LeuT proteoliposomes ................................................................... 107 Transport assays .................................................................................................... 108 Binding assays ........................................................................................................ 108 Crystallization, data collection, model refinement ................................................. 109 Results and Discussion ........................................................................................... 110 LeuT-TnaT sequence alignment suggests altered binding site within a conserved structure ................................................................................. 110 iii TnaT homology model suggests binding site substiutions are specificity-altering ........................................................................................... 111 I359Q substitution in LeuT confers tryptophan transport activity to LeuT ........ 113 F259V does not inhibit binding of trp to LeuT ...................................................... 114 Steady-state kinetics of trp transport by LeuTI359Q............................................... 114 Trp transport active LeuT mutants bind trp in the occluded state...................... 114 The Trp602 site is absent in the Trp-bound occluded state ................................... 116 A structural description of the transport cycle ..................................................... 117 Tables ........................................................................................................................ 119 Figures ....................................................................................................................... 120 Chapter 5 Architectures and Archetypes of Sodium-Coupled