Highways, biways, and detours: the IspD story

Transcription

1 Highways, biways, and detours: the IspD story RAMC September Terese Bergfors

2 Sweden

3 Linnaeus Celsius Ångström

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5 RAPID: Rational Approaches to Pathogen Inhibitor Discovery

6 How to choose a target? Essentiality studies Homology Important biochemical pathways

7 How to choose a target? Essentiality studies Homology M. smegmatis, M. tuberculosis Important biochemical pathways

8 How to choose a target? Essentiality studies Homology Important biochemical pathways MEP (a non-mevalonate pathway) for isoprenoid synthesis

9 Outline of Crystallization Strategy What initial screen to use? What to do if you get a hit What to do if you don t get a hit with our real-life example IspD

10 Our setup Cloning His-His-His-His-His-His Expression Arabinose liter Purification Nickel IMAC Superdex

11 The initial search problem: how many conditions to try?

12 Is minimal screening efficient? # conditions Results N= more 21 Newman (2005) Acta Cryst D61

13 How similar/different are two screens? A metric for: The C6 Web Tool at CSIRO C3 internal diversity diversity between screens search for a particular chemical

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16 bis-trispropane ph 6.5

17 Examples of start buffers RAPID, Uppsala JCSG, California SECSG, Georgia Toronto, Canada Tris ph 7.5, NaCl, 10% glycerol Tris ph 7.9, NaCl, 0.25 mm TCEP Tris, ph 7.5, NaCl, 0.1 mm protease inhibitor mix Hepes, 7.5, NaCl

18 Optimum Solubility Buffer Screen Problem 14 proteins with poor solubility Action Result Screen for better buffer conditions---improved solubility for 11 of them 9 of them now crystallized Jancarik et al. Acta Cryst 2004 D60,

19 Optimum solubility buffer screen How to do it: Google these words: Optimum Solubility Screen SOP also: Kits available Thermofluor Jancarik et al. Acta Crys D60, 2004

20 After the buffer change from Hepes, 7.5 to btp, 6.5

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22 Outline How many conditions to screen? What to do if you get a hit What to do if you don t get a hit

23 screening optimization

24 Two different search problems Initial hit Optimization grid search seeding ligands/substrates additive screens change kinetics

25 0.3 mm Screening Optimization with CTP

26 Dataset collected on MAR images x 15 min exposure = 45h collection time. Resolution: 2.2Å Spacegroup: p

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28 Outline How many conditions to screen? What to do if you get a hit What to do if you don t get a hit

29 Run SDS gel Minimal screens no yes optimize Rescue strategies no Screen more yes

30 Check the protein concentration! Minimal screens no yes optimize Rescue strategies no Screen more yes

31 3 mg/ml or 40 mg/ml? Initial Screening Optimized with ligand 0.3 mm

32 What salvage method to use? What rescue method to use? in situ proteolysis reductive methylation truncations remove disordered regions use an ortholog:cross-seed

33 M. smegmatis IspD + CTP M. smegmatis IspD + CMP M. tuberculosis IspD + CTP Björkelid, Bergfors et al Acta D 2011

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35 The IspD Story: Summary Protein concentration A truncation Change of buffer Add substrate Good teamwork

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37 xray.bmc.uu.se/terese

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39 Predicting crystallizability Dynamic light scattering (DLS) Bioinformatics Thermofluor Calorimetry Mass spec NMR

40 Predicting crystallizability vs. predicting disorder XtalPred PDPredictor MetaPPCP ParCrys OB-Score, etc.

41 TCA precipitation test

42 TCA precipitation test 0.5 mg/ml 2.5 mg/ml 5.0 mg/ml

43 Example of conformational heterogeneity Cellobiohydrolase digesting a chain of

44 Why is it so hard to crystallize proteins? Evolution is against it! Crystallization conditions are unique for each protein. Proteins can be unstable, different conformations, floppy, etc.

45 DLS after freeze/thaw Grey: before freezing Black:after thawing

46 Stability Test at 4 and 20 o C Reduced Nonreduced

47 Two analyses of disorder Glob Plot Fold Index

48 Not much consensus! Fold Index Glob Plot RONN IU Pred POND R Fold Unfol Prelink Disprot Disopred Fold Index Glob Plot RONN Prelink Disprot IU Pred Pondr Fold Unfol 62

49 Overlap of methods Vapor-diffusion hanging-drop

50 Overlap of methods Vapor-diffusion hanging vs. sitting

51 Overlap of methods 70% Vapor-diffusion Microbatch

52 Overlap of methods Vapor-diffusion Microbatch Counterdiffusion

53 Matrix Microseeding MMP12 BVP USP7 Trypsin PPE D Arcy et al. Acta Cryst. (2007). D63