Structural Study of the Acid Sphingomyelinase Protein Family Alexei Gorelik Department of Biochemistry McGill University, Montreal August 2017 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Alexei Gorelik, 2017 Abstract The acid sphingomyelinase (ASMase) converts the lipid sphingomyelin (SM) to ceramide. This protein participates in lysosomal lipid metabolism and plays an additional role in signal transduction at the cell surface by cleaving the abundant SM to ceramide, thus modulating membrane properties. These functions are enabled by the enzyme’s lipid- and membrane- interacting saposin domain. ASMase is part of a small family along with the poorly characterized ASMase-like phosphodiesterases 3A and 3B (SMPDL3A,B). SMPDL3A does not hydrolyze SM but degrades extracellular nucleotides, and is potentially involved in purinergic signaling. SMPDL3B is a regulator of the innate immune response and podocyte function, and displays a partially defined lipid- and membrane-modifying activity. I carried out structural studies to gain insight into substrate recognition and molecular functions of the ASMase family of proteins. Crystal structures of SMPDL3A uncovered the helical fold of a novel C-terminal subdomain, a slightly distinct catalytic mechanism, and a nucleotide-binding mode without specific contacts to their nucleoside moiety. The ASMase investigation revealed a conformational flexibility of its saposin domain: this module can switch from a detached, closed conformation to an open form which establishes a hydrophobic interface to the catalytic domain. This open configuration represents the active form of the enzyme, likely allowing lipid access to the active site. The SMPDL3B structure showed a narrow, boot-shaped substrate binding site that accommodates the head group of SM. However, no in vitro lipid hydrolysis could be detected; further work is required to identify potential bona fide substrates. In summary, these studies illustrate how an enzyme family can adapt a conserved architecture and mechanism to perform divergent functions. 2 Résume La sphingomyélinase acide (ASMase) produit le lipide céramide à partir de la sphingomyéline (SM). Cette protéine contribue au métabolisme des lipides dans le lysosome et participe à la transduction de signaux sur la membrane plasmique en altérant les propriétés de cette dernière par son action. Ces fonctions sont accomplies avec l’aide du domaine saposine de cette enzyme, qui interagit avec les lipides et les membranes. ASMase fait partie d’une famille de protéines dont les deux autres membres sont mal caractérisés : les phosphodiestérases SMPDL3A et B. SMPDL3A n’hydrolyse point la SM mais dégrade par contre les nucléotides extracellulaires, ce qui implique potentiellement cette enzyme dans la signalisation purinergique. SMPDL3B régule les réponses immunitaires innées ainsi que le fonctionnement des podocytes par l’entremise d’une activité incomplètement définie de modification de lipides et de membranes. J’ai mené des études structurales sur cette famille de protéines afin de mieux comprendre leurs fonctions moléculaires et la reconnaissance des substrats par ces enzymes. Les structures cristallines de SMPDL3A ont révélé le repliement hélicoïdal d’un nouveau sous-domaine C- terminal, un mécanisme catalytique légèrement modifié, et un mode de liaison aux nucléotides dénué de contacts spécifiques avec leur portion nucléoside. L’investigation de l’ASMase a démontré la flexibilité conformationnelle de son domaine saposine : ce module alterne entre une configuration fermée détachée, et une forme ouverte qui interagit avec le domaine catalytique via une interface hydrophobe. Cette conformation ouverte représente la forme active de la protéine, probablement en facilitant l’accès des lipides au site actif. La structure de SMPDL3B a dévoilé un site de liaison au substrat étroit en forme de botte, où le groupe de tête de la SM peut se positionner. Par contre, aucune hydrolyse de lipides n’a pu être démontrée in vitro; des travaux additionnels sont requis afin d’identifier des substrats authentiques. En résume, ces études illustrent la capacité d’une famille d’enzymes à adapter une structure et un mécanisme communs vers des fonctions variées. 3 Table of contents Abstract 2 Résumé 3 Table of contents 4 List of figures 7 List of tables 9 List of abbreviations 10 Preface 12 Author contributions 13 Acknowledgements 14 Chapter 1 – General introduction 15 1.1 The calcineurin-like phosphoesterases 15 1.1.1 Shared structural features 17 1.1.2 PPP-type phosphatases 20 1.1.3 Other serine/threonine phosphatases 20 1.1.4 Nucleases 21 1.1.5 Nucleotidases 21 1.1.6 Enzymes of other or unknown function 22 1.1.7 Bacterial enzymes 23 1.1.8 Acid sphingomyelinase-like proteins 23 1.2 Acid sphingomyelinase 24 1.2.1 Sphingolipids 24 1.2.2 Sphingomyelinases 26 1.2.3 Niemann-Pick disease types A and B 27 1.2.4 Lysosomal roles of ASMase 28 1.2.5 Saposins 29 1.2.6 Roles of ASMase at the cell surface 32 1.2.7 ASMase in the circulation 34 1.3 SMPDL3B 34 4 1.3.1 Lipid rafts in toll-like receptor signaling 35 1.3.2 ASMase in TLR signaling 35 1.3.3 SMPDL3B in TLR signaling 36 1.3.4 Lipid rafts in podocytes 38 1.3.5 SMPDL3B in podocyte function 38 1.3.6 SMPDL3B outside of the cell 40 1.4 SMPDL3A 41 1.4.1 SMPDL3A in the cholesterol response 41 1.4.2 Purinergic signaling 42 1.5 Conclusion 43 Chapter 2 – Study of SMPDL3A 45 Introductory transition 45 2.1 Abstract 46 2.2 Introduction 47 2.3 Results 49 2.4 Discussion 65 2.5 Experimental procedures 67 Concluding transition 69 Chapter 3 – Study of ASMase 74 Introductory transition 74 3.1 Abstract 75 3.2 Introduction 76 3.3 Results 78 3.4 Discussion 103 3.5 Experimental procedures 105 Concluding transition 109 Chapter 4 – Study of SMPDL3B 116 Introductory transition 116 5 4.1 Abstract 117 4.2 Introduction 118 4.3 Results 121 4.4 Discussion 134 4.5 Experimental procedures 138 Concluding transition 141 Chapter 5 – Conclusion 144 References 148 6 List of figures Figure 1.1 Mammalian members of the calcineurin-like phosphoesterase superfamily 16 Figure 1.2 Structural superimposition of calcineurin-like phosphoesterases 18 Figure 1.3 Active site of calcineurin-like phosphoesterases 19 Figure 1.4 Chemical structures of representative lipid species 25 Figure 1.5 Structural flexibility of saposins 31 Figure 2.1 Sequence alignment of SMPDL3A and ASMase 50 Figure 2.2 Structure of SMPDL3A 52 Figure 2.3 Active site of SMPDL3A 54 Figure 2.4 Inhibition of SMPDL3A by excess zinc 56 Figure 2.5 Ligands bound in the active site of SMPDL3A 58 Figure 2.6 Proposed reaction mechanism for SMPDL3A 61 Figure 2.7 Nucleotide binding modes of human and murine SMPDL3A 70 Figure 3.1 Structural overview of ASMase 79 Figure 3.2 The ASMase fold 81 Figure 3.3 Comparison and mutation of the ASMase active site and proposed catalytic mechanism 84 Figure 3.4 ASMasesap – ASMasecat interactions 87 Figure 3.5 Purification of murine ASMase and bNPP hydrolysis assay 90 Figure 3.6 Purification of human ASMase and activity assays 92 Figure 3.7 Main chain B-factor analysis of catalytic domain interface loops 94 Figure 3.8 Saposin dimers in the crystal lattice 96 Figure 3.9 Electrostatic surface and substrate binding site of ASMase 97 Figure 3.10 Liposomal activity assay of ASMase in the presence of cationic amphiphilic drugs (CADs) 98 Figure 3.11 Electron density and binding mechanism for the co-crystallized inhibitor AbPA 100 Figure 3.12 Structural mapping of disease mutations 102 Figure 3.13 Schematic for ASMase activation 104 7 Figure 3.14 Representative electron density maps of the ASMase structures 108 Figure 3.15 PC recognition by human ASMase 110 Figure 3.16 Lipid delivery to GALC by SapA 112 Figure 4.1 Crystal structure of SMPDL3B 122 Figure 4.2 Structural comparison of SMPDL3B, SMPDL3A and ASMase 124 Figure 4.3 Region around active site 126 Figure 4.4 Phosphocholine bound in the active site of SMPDL3B 128 Figure 4.5 In vitro enzymatic activity against various substrates 130 Figure 4.6 Functional impact of SMPDL3B mutants on LPS-induced IL-6 release in macrophages 133 Figure 4.7 Proposed binding modes of potential substrates 136 Figure 4.8 Putative rituximab epitope on SMPDL3B 142 8 List of tables Table 2.1 SMPDL3A X-ray data collection and structure refinement statistics 71 Table 2.2 Michaelis-Menten parameters of ATP hydrolysis by SMPDL3A 73 Table 3.1 ASMase X-ray data collection and structure refinement statistics 78 Table 3.2 Predicted effects of ASMase mutations found in Niemann-Pick patients 101 Table 3.3 Predicted effects of ASMase variants of unknown significance 115 Table 4.1 SMPDL3B X-ray data collection and structure refinement statistics 143 9 List of abbreviations AbPA – 1-aminodecylidene bis-phosphonic acid alk-SMase – alkaline sphingomyelinase AMPCPP – ɑ,β-methylene ATP AP4A – diadenosine tetraphosphate ASMase – acid sphingomyelinase BMP – bis(monoacylglycero)phosphate bNPP – bis(4-nitrophenyl) phosphate CAD – cationic amphiphilic drug CTD – C-terminal domain / subdomain DKD – diabetic kidney disease E-NPP – ecto-nucleotide pyrophosphatase / phosphodiesterase E-NTPD – ecto-nucleoside triphosphate diphosphohydrolase ERT – enzyme replacement therapy FSGS – focal segmental glomerulosclerosis GPI – glycosylphosphatidylinositol IL-6 – interleukin-6 LDL – low density lipoprotein LPS – lipopolysaccharide M6P – mannose-6-phosphate NP – Niemann-Pick NPPC – p-nitrophenylphosphorylcholine PC – phosphocholine pNP-PC – p-nitrophenyl phosphorylcholine pNP-TMP – p-nitrophenyl thymidine 5'-monophosphate SM – sphingomyelin SMase – sphingomyelinase SMPD1 – sphingomyelin phosphodiesterase 1 SMPDL3A – acid sphingomyelinase-like phosphodiesterase 3a SMPDL3B – acid sphingomyelinase-like phosphodiesterase 3b 10 suPAR – soluble urokinase-type plasminogen activator TLR – toll-like receptor TM – transmembrane TNFα – tumor necrosis factor α uPAR – urokinase-type plasminogen activator 11 Preface This is a manuscript-based thesis consisting of three published articles [211,221,228], in chronological order: Chapter 2 Gorelik A, Illes K, Superti-Furga G, Nagar B.