05/05/2011 Exploring components of the innate immune system in a combined SAXS, NMR and crystallography approach. Haydyn Mertens PhD
The SAXS experiment Elastic & coherent scattering only considered here. Wave vector k Detector Sample: Dilute system of particles k 0 k 0 s k 1 Monochromatic beam source: X-ray tube (0.1 -0.2 nm) Synchrotron (0.05 - 0.5 nm) Thermal neutrons (0.1 - 1 nm) Scattering vector s
SAXS in modern structural biology Mertens & Svergun, J Struct Biol. 2010 Oct;172(1):128-41
Modular systems
Modular systems • Molecules composed of modular building blocks o functional modules o structural modules • Can be intrinsically flexible • Hard to crystallize entire system • NMR spectra become complex • Divide and conquer approach suitable
Example: FactorH FactorH is a 155 kDa plasma glycoprotein (1213 aa) Contains 20 CCP modules (60 aa) (CCP = Complement Control Protein) C3b interaction sites Polyanion binding sites FactorH is involved in regulation of complement activation
Complement Activation • The complement system regulates: – Self vs non-self recognition – Via soluble and cell-surface regulators – Prevention of damage to host tissue – Target “tag” foreign bodies • Most regulatory proteins act on convertases: – C3ase (C3 convertase) – C5ase (C5 convertase)
Complement Activation Classic pathway C1 + Antigen-Antibody C3ase + C5,C6,C7,C8,C9 C3 C3b Cell Death C3ase C5ase FactorD FactorB Alternative pathway C3 C3b
Regulation of Complement Activation Regulation of self VS non-self surface recognition FactorH binds C3b and C3ase FactorH + C5,C6,C7,C8,C9 C3 C3b Cell Death C3ase C5ase FactorD FactorB FactorH Alternative pathway C3 C3b
Regulation of Complement Activation • C3ase activity is inhibited: FactorH-C3ase • C3b targeted for degradation: FactorH-C3b + C5,C6,C7,C8,C9 C3 C3b Cell Death C3ase C5ase FactorD FactorB Alternative pathway C3 C3b
Regulation of Complement Activation FactorH binds more effectively to C3ase and C3b associated with HOST surfaces. Enhanced by the presence of polyanionic markers (eg. glycosaminoglycans) FactorH C3b C3ase Host membrane surface
Atyplical hemolytic uremic syndrome • aHUS caused by mutations in Factor-H – Thus defective Self vs non-self recognition • Disease leads to: – Hemolytic anemia – Renal failure – Death http://www.arizonatransplant.com
FactorH • BIG QUESTION: o How does FactorH interact with C3b/C3ase? • Start with: o Determine CCP structures (NMR/X-ray) o What are the possible interaction modes? o Are the central CCP modules structural rather than functional? o Flexible or rigid structure? • Then: study the C3:FactorH complex!
FactorH: High-res structures 15-16 6-8 7 19-20 (NMR) 19-20 (X-ray) 1-2 2-3
FactorH-C3b partial structure Crystal structures map part of the interaction interface: C3b(a) C3b(b) FH 1-4 Wu et al. (2009), Nat. Immunol., 10:728-33.
FactorH: ccp19 & ccp20 FH 19-20 titrated with C3. Lineshape changes indicate: • C3 contacts FH-19 • Small contact with FH-20 FH 19-20 titrated with polyanions. Chemical shift changes indicate: • FH-20 binds polyanions Morgan et al., Nat Struct Mol Biol. 2011 Apr;18(4):463-70.
FactorH binding mode question? • CCPs 1-4 and CCPs 19-20 interact with C3b. • CCP 20 interacts with surface polyanions • Bent back structure expected: – Allows for a bidentate binding mode. – Is the core flexible/rigid?
SAXS studies on free FactorH
FactorH: Possible configurations Extended (Dmax ~ 75 nm) Horse-shoe Bent-back
Factor H: Central core 10-15 • CCP's 10-15 may form a structural core • Bent back or horeshoe structure for C3b interaction? • Linkers provide flexibility? Rigid structure?
Factor-H: information from SAXS • What information can we get? o Overall size and shape (Rg, Dmax, MM) o Low resolution ab initio models o Higher resolution rigid body models (using known sub-structures) o Characterisation of flexibility
Quick note on sample preparation • Sample prep essentially identical for SAXS & NMR • A good NMR sample should be good for SAXS Concentration series essential for identifying inter-particle interactions (eg. aggregation, repulsion) 10, 5, 2, 1 mg/ml usually fine (for proteins > 10 kDa) BEWARE radiation damage!!! add DTT or glycerol for scavenging free radicals
Radiation Damge: eg. GST • GST +/- 2.5 % (v/v) glycerol Significant increase in intensity at low angles
Info directly from SAXS data:
Info directly from SAXS data: • Overall parameters determined from the curves • Size o MM (from I(0) ), o hydrated volume, Vp • Shape/Dimension o Rg , Dmax o Distance-distribution (GNOM)
Info directly from SAXS data: • Guinier analysis o MM (from I(0) ) o Rg ln[I] = ln[I(0)] -1/3(Rg*s) 2
Info directly from SAXS data: • Distance-distribution, P(r) o MM (from I(0) ) o Rg o D m a x o V p
Overall SAXS parameters: Factor-H Construct Rg , nm Dmax , nm MM , kDa Vp , nm3 ( MM , kDa) FH 10-15 3.1 10.4 46 68 (41.1) FH 11-14 3.1 10.5 35 38 (27.3) FH 10-12 2.7 8.9 26 37 (20.6) FH 12-13 2.2 7.1 13 20 (13.6) For the Factor-H constructs these parameters, extracted within minutes of measurement tell us that the core is compact!
Calculation of scattering curves
Calculation of scattering curves • CRYSOL (Svergun et al., 1995) Aa - Atomic scattering in vacuo As - Scattering from the excluded volume Ab - Scattering from the hydration shell
Validation of solution structure • NMR structures determined: o FH 11-12 , FH 12-13 • NMR structure in progress: o FH 10-11 Schmidt et al. (2010), J. Mol. Biol., 395:105-22.
Validation of solution structure • Fitting NMR conformers ( 11-12 , 12-13 ) to SAXS data o CRYSOL (Svergun et al., 1995)
Hand built model: Fh10-12 • Using overlapping NMR models o Fit to SAXS data (no refine) CCP11 CCP10 CCP12 • Fit looks good o Use Fh10-12 model as single rigid body for larger constructs
Hand built model: Fh10-12 • Using overlapping NMR models o Fit to SAXS data (no refine) SAXS envelope CCP11 CCP10 CCP12 • Fit looks good o Use Fh10-12 model as single rigid body for larger constructs
Ab initio modelling: Factor-H • Shape envelopes determined from SAXS data alone o DAMMIF (Franke & Svergun, 2009) Fh 11-12 6.5 nm Fh 12-13 7.1 nm
Ab initio modelling: Factor-H • Larger constructs Fh 10-15 10.4 nm Fh 11-14 Fh 10-12 10.5 nm 8.9 nm Schmidt et al. (2010), J. Mol. Biol., 395:105-22.
Ab initio modelling: Factor-H • The ab initio models provide: o Indication of compaction for Fh10-15 o Compact end for Fh10-15? o Fh11-14 suggests zig-zag bent core • Not consistent with a horse-shoe shape core • Low resolution but useful information!
Rigid body modelling
Rigid body modelling Rigid body modelling of complexes • Finds optimal configuration of subunits in a complex o I(s) calculated using spherical harmonics o Multiple curves, contrasts & symmetry supported o User-defined contacts & orientation constraints o Minimises steric clashes Petoukhov, M. V. & Svergun, D. I. (2005). Biophys. J. 89 , 1237-1250
Rigid body modelling • Use additional structural data to improve resolution of modelling • Using known CCP structures o SASREF o BUNCH/CORAL (models linkers) • Using overlapping NMR models o Low temperature refinement o Decrease possible solutions
Rigid body modelling: Fh11-14 • Using Fh11-12 and single CCPs o CCP13 overlaid as in NMR structure. o CCP14 free to move o Low temperature refinement CCP12 CCP13 CCP14 CCP11
Rigid body modelling: Fh10-15 • CCPs 10-13 fixed o Good fit but CLASHES • Free CCP10 o Good fit no clashes CCP10 free o Flexible 10-11 linker? CCP13 CCP14 CCP10 10-13 fixed CCP11 CCP15 CCP12 • NOT a horse-shoe shaped structure
The Factor-H core is compact: • Addition of CCPs 10 and 15 do not increase overall dimension relative to Fh11-14 • CCPs 12-13 form a kink in the structure • CCPs 13,14,15 form a compact terminus • Segment 10-15 may allow for a "bending-back" • Is the structure flexible?
Assessment of flexibilty with ensembles
Flexibility analysis by EOM • Ensemble optimisation method (EOM) o SAXS data driven selection from a random pool P. Bernado, E. Mylonas, M. V. Petoukhov, M. Blackledge & D. I. Svergun (2007) JACS, 129, 5656-5664
Flexibility analysis by EOM • Ensemble optimisation method (EOM) o SAXS data driven selection from a random pool • How does the program work? o Generation of random pool of models. o Genetic algorithm. o Best ensemble selected. o Provides Rg & Dmax distributions P. Bernado, E. Mylonas, M. V. Petoukhov, M. Blackledge & D. I. Svergun (2007) JACS, 129, 5656-5664
Flexibility analysis by EOM • eg. Poly-ubiquitin o data (single structure) o data (flexible case) • Rg distributions o Broad = flexible o Narrow = "rigid" P. Bernado,(2010), Eur Biophys. J 39(5) 769-80
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