Structural Studies of Human GBE1 and Relevance to APBD


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Structural Studies of Human GBE1 and Relevance to APBD

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  • Compartment (pH)Concentration
  • Compartment (pH)Concentration
  • No specific cure, diet. Vitamin B6 can reduce some eye bone, clotting problem
  • Structural Studies of Human GBE1 and Relevance to APBD

    1. 1. Structural studies of human GBE1 and relevance to APBD Wyatt Yue Structural Genomics Consortium (SGC) University of Oxford APBDRF Meeting Dec 2013
    2. 2. • Public-private partnership • High-throughput structural & chemical biology of human proteins • Diverse protein families/biology areas • Open access research model Combining structural biology, protein biochemistry to understand inborn errors of metabolism
    3. 3. Studying enzyme shapes • Enzymes are proteins - building blocks (amino acids) - have different shapes • Shapes can change too! • Understanding enzyme functions/malfunctions – need to know its shapes … seeing molecules in action
    4. 4. ‘Taking pictures’ of protein structures 1. make proteins of interest (expression, purification) 3. take picture! (x-ray diffraction) 2. arrange them in order (crystallization) 4. develop the film (modelling)
    5. 5. Our work so far … APBDRF Meeting Dec 2013
    6. 6. Glycogen Synthesis human GYG1 structure priming glycogenin GSD type XV elongation Cartoon impression of glycogen granule glycogenin glycogen synthase GSD type 0 branching glycogenin human GBE1 structure branching enzyme GSD type IV (liver) Adult Polyglucosan Body Disease glycogen synthase
    7. 7. Multi-construct approach 1 700 702 E coli structure 63 63 st round 1 70 Insoluble in E. coli 70 79 79 2nd round Soluble in insect cells 3rd round Fine-tune c000 c001 c002 c003 c004 c005 c101 c102 c103 c104 c105 c106 c107 c108 1 1 16 16 38 38 54 54 L28 P36 Y41 R47 c109 c110 c111 c112 c011, c012 c013, c014 c015, c016 c017, c018 • • • Full length and a series of truncations N- and C-termini nibbling N-terminal His6 tagged fusion
    8. 8. GBE1 Purification & Crystallization GBE1A-c014 (recombinant Pro37-Leu700 with a TEV-cleavable His6-tag) Growth from 4L of Baculovirus-infected insect Sf9 cells 2. Size exclusion 1. Ni affinity 250 FT BB1 WB1 E1 E3 BB2 WB2 E2 150 3. His-tag removal by TEV 4. Second Ni affinity (to remove uncut protein, TEV) GBE1A c011 E1 s200 GF 300812:Sample1Title_UV GBE1A c011 E1 s200 GF 300812:Sample1Title_Logbook GBE1A c011 E1 s200 GF 300812:Sample1Title_Fractions u/c GBE1A c011 E1 s200 GF 300812:Sample1Title_Inject mAU 350 Pooled Pooled 200 Pooled 150 Stop Collecting Fractions 15 Start Collecting Fractions Sample 1 Name 50 Finish Washing Fraction Collector Outlet 100 Wash Fraction Collector Outlet 25 20 BB1 WB GF BB2 EB 250 75 37 FT 300 100 50 cut 0 10 A2 0 A4 A6 20 A7 40 A9 A11 B12 B10 B8 B6 60 B4 B2 C1 C3 C5 C7 80 C9 C11 D12 D10 D8 100 D6 D4 D2 E1 E3 E5 120 E7 E9 E11 F12 F10 F8 140 ml Concentrated, purified 15% PEG 3350 0.15M sodium succinate
    9. 9. Structure determination • Three structure data: – apo GBE1, GBE1-acarbose and GBE1-Glc7 Complex with Overall structure sugar chains Mapping of gbe1 mutations Y329 site
    10. 10. GBE1 mutants 1 702 38 700 c106 Y329S R515H R524Q D357A E412A DISEASE CATALYTIC nucelophile acid/base catalyst 1 L insect cells, standard purification procedure Y329S R515H R524Q D357A Majority are expressed but only p.D357A is soluble. Attempts were made to optimise the purification of p.Y329S. E412A
    11. 11. p.Y329S mutant protein purification Scale up from 6L insect cell culture Ni affinity Total Soluble FT WB1 WB2 WB3 WB4 WB5 Test TEV Cleavage E1 250 150 100 75 50 37 50 37 25 20 15 25 20 15 GBE1A p.Y329S band confirmed by tryptic digestion. GF, concentrated uncut cut E2 250 150 100 75 Concentrated GF Samples 250 150 100 75 50 37 25 20 15 GBE1A-c201 e129 p014 p015 (6L, Baculo)
    12. 12. Pharmacological Chaperones Many metabolic disorders are misfolding defects Pharmacological Chaperone (PCs) Rescue of stability and function Examples of PC development Disease Protein Chaperone Fabry GLA DGJ Gaucher GBA IFG, DNJ GM1 GLB1 NOEV GM2 HEXA Pyrimethamine Pompe GAA DNJ MPSIII NAGLU 2AcDNJ, 6AcCAS Fan et al. 1999; Yu et al. 2007; Matsuda et al. 2003; Maegawa et al. 2007; Parenti et al. 2007;Ficko-Blean et al. 2008 Yet, their action is counter-intuitive: They compete with native substrate/cofactor Strategy: Develop non-competitive, allosteric PCs?
    13. 13. Next Action • Scale up less soluble proteins – WT full-length protein – Mutants Y329S, R515H, R524Q Different tags/fusion proteins to improve solubility • Characterize hits/peptides (Tropak/Kakhlon) – DSF, limited proteolysis – Aggregation/unfolding assay – co-crystallization, ITC Time, protein consuming
    14. 14. Future: collaboration with APBD APBDRF Meeting Dec 2013
    15. 15. Working with rare disease patient groups Inborn errors of metabolism Rare cancers & developmental disorders Membrane proteins & rare diseases Genomic variation & disease Alex Bullock ‘Stone man Collaborative projects syndrome’ Funded personnel Public engagement Information
    16. 16. What can we do to help APBDRF? Clinical ‘know-how’ WT/mutant protein Crystals, 3D Structure hit finding xtal soaking in silico Docking HT Compound Screening (Michael Tropak) in silico Ligand Design (peptides – Or Kakhlon) Binding, Biochemistry DSF, ITC, BLI, co-xtal dose response, affinity validation Cellular Assays Mode of Action folding, proteolysis, aggregation Effects on activity, stability Test Mutants rescue stability? Effects on activity characterization patient fibroblast cells Animal model (e.g. mouse) enzyme activity Rescue in vivo? Goal: To develop compounds into a pharmacological chaperone treatment
    17. 17. Example of our In Vitro capabilities Pathfinder Awards for Orphan Diseases Cystathionine beta synthase (CBS) deficiency Aim: look for binders at different pockets/regions as chemical starting point human CBS structure Allosteric domain (AdoMet) Recombinant protein Structural Biology Catalytic domain (PLP, haem) Fragment DSF Compound library Cellular assay Structurally diverse, drug-like hits Developed In vitro assays to deconvolute binding modes In silico docking Limited proteolysis kp = 0.381 ± 0.037 min-1 Domain mapping
    18. 18. ACKNOWLEDGEMENTS THE MOB 2013 Thomas McCorvie Dipali Patel Jolanta Kopec Stephanie Oerum Fiona Fitzpatrick Sean Froese Wasim Kiyani CRYSTALLOGRAPHY Frank von Delft Tobias Krojer 2012 BIOTECHNOLOGY Claire Damerell Pravin Mahajan FUNDING PARTNERS The Canadian Institutes for Health Research, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Lilly Canada, the Novartis Research Foundation, Pfizer, Takeda, the Ontario Ministry of Economic Development and Innovation, and the Wellcome Trust.
    19. 19. Structure determination • Three structure data: – apo GBE1, GBE1-acarbose and GBE1-Glc7 Complex with Overall structure sugar chains Mapping of gbe1 mutations Y329 site
    20. 20. Updates on DSF APBDRF Meeting Dec 2013
    21. 21. Thermal stability as a ligand binding assay Differential Scanning Fluorimetry (DSF), ‘Tm shift’ WT Small molecule stabilization? – right shift towards WT Niesen et al 2007 Nat Methods Stabilization by native ligands – right shift (+ Tm) Destabilization due to mutation – left shift (- Tm)
    22. 22. DSF considerations for GBE1 No detectable Tm shift with various sugar analogues N=2 experiments - Even at higher concentrations as tested previously - despite electron density in structures! - These are not natural ligand but shortened versions Tm for apo protein varies with experiments - error margins large, with experiments/preps - is stability concentration dependent? Is conformational change required for catalysis? (This increases likelihood of seeing Tm shift) - Compounds may not be ‘large’ enough a? Difference between Full-length vs truncated proteins (previous) E. coli expressed, FL protein - Tm varies with constructs - ->N-terminal may be important for stability/disease? - may explain lack of detectable binding? Tm = 41.8 ± 2.1 °C N=8 experiments Acarbose (4) Glc4,Glc7 4-mer peptides
    23. 23. GBE1 constructs purified 1 c105 c106 c011 c013 c014 c015 38 38 28 36 36 41 p001 = cut c105  used p002 = cut c106  used p003 = cut c106  used p004 = uncut c106  ~200ul 16.5mg/ml p005 = cut c106  ~100ul 2.2mg/ml p006 = cut c011 ~120ul 6.9mg/ml 02 Oct 2012, 15 Jan 2013 *p007 = cut c014  ~530ul 16.1mg/ml *p009 = uncut c013  ~550ul 20.2mg/ml 15 Sep 2013 p010 = cut c013  ~330ul 3.5mg/ml p011 = uncut c015  ~250ul 7.3mg/ml p012 = cut c015  ~80ul 7.3mg/ml p013 = uncut c106  ~50ul 10.7mg/ml 700 702 702 700 702 702 700 702
    24. 24. Structural analysis of Y329 site WT p.Y329S Y329 forms hydrogen bond between its side-chain and backbone carbonyl of His289. 2.5 Å distance in WT increases to 8.5 Å in p.Y329S. Virus re-amplified , extraction buffer optimised to sodium phosphate based, Talon (cobalt) used instead of Ni-NTA (Nickel).
    25. 25. Structure determination • Three structure data: – apo GBE1, GBE1-acarbose and GBE1-Glc7