Exploring components of the innate immune system in a combined SAXS, - - PowerPoint PPT Presentation

05/05/2011

Exploring components of the innate immune system in a combined SAXS, NMR and crystallography approach.

Haydyn Mertens PhD

Elastic & coherent scattering only considered here.

The SAXS experiment

k0 k0 k1 s

Sample: Dilute system of particles

Detector

Monochromatic beam source: X-ray tube (0.1 -0.2 nm) Synchrotron (0.05 - 0.5 nm) Thermal neutrons (0.1 - 1 nm)

Wave vector k

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
• functional modules
• 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 C3 C3b C3ase C5ase Cell Death

+ C5,C6,C7,C8,C9

C1 + Antigen-Antibody C3ase C3 C3b FactorB FactorD Alternative pathway

Regulation of Complement Activation

C3 C3b C3ase C5ase Cell Death

+ C5,C6,C7,C8,C9

C3 C3b FactorB FactorD Alternative pathway Regulation of self VS non-self surface recognition FactorH binds C3b and C3ase FactorH FactorH

Regulation of Complement Activation

C3 C3b C3ase C5ase Cell Death

+ C5,C6,C7,C8,C9

C3 C3b FactorB FactorD Alternative pathway

• C3ase activity is inhibited: FactorH-C3ase
• C3b targeted for degradation: FactorH-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:
• How does FactorH interact with C3b/C3ase?
• Start with:
• Determine CCP structures (NMR/X-ray)
• What are the possible interaction modes?
• Are the central CCP modules structural rather than

functional?

• 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: FH 1-4 C3b(a) C3b(b)

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

• CCP's 10-15 may form a structural core
• Bent back or horeshoe structure for C3b interaction?
• Linkers provide flexibility? Rigid structure?

Factor H: Central core 10-15

Factor-H: information from SAXS

• What information can we get?
• Overall size and shape (Rg, Dmax, MM)
• Low resolution ab initio models
• Higher resolution rigid body models (using known

sub-structures)

• 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

• GST +/- 2.5 % (v/v) glycerol

Significant increase in intensity at low angles

Radiation Damge: eg. GST

Info directly from SAXS data:

Info directly from SAXS data:

• Overall parameters determined from

the curves

• Size
• MM (from I(0)),
• hydrated volume, Vp
• Shape/Dimension
• Rg, Dmax
• Distance-distribution

(GNOM)

Info directly from SAXS data:

• Guinier analysis
• MM (from I(0))
• Rg

ln[I] = ln[I(0)] -1/3(Rg*s)2

Info directly from SAXS data:

• Distance-distribution, P(r)
• MM (from I(0))
• Rg
• Dm

a x

• Vp

Overall SAXS parameters: Factor-H

For the Factor-H constructs these parameters, extracted within minutes of measurement tell us that the core is compact!

Construct (MM, kDa) Rg, nm Dmax, nm MM, kDa Vp, nm3 FH 10-15 (41.1) 3.1 10.4 46 68 FH 11-14 (27.3) 3.1 10.5 35 38 FH 10-12 (20.6) 2.7 8.9 26 37 FH 12-13 (13.6) 2.2 7.1 13 20

Calculation of scattering curves

Calculation of scattering curves

• CRYSOL (Svergun et al., 1995)

As - Scattering from the excluded volume Aa - Atomic scattering in vacuo Ab - Scattering from the hydration shell

• NMR structures determined:
• FH 11-12, FH 12-13
• NMR structure in progress:
• FH 10-11

Schmidt et al. (2010), J. Mol. Biol., 395:105-22.

Validation of solution structure

Validation of solution structure

• Fitting NMR conformers (11-12, 12-13) to SAXS data
• CRYSOL (Svergun et al., 1995)

Hand built model: Fh10-12

• Using overlapping NMR models
• Fit to SAXS data (no refine)

CCP10 CCP11 CCP12

• Fit looks good
• Use Fh10-12 model as single rigid

body for larger constructs

Hand built model: Fh10-12

• Using overlapping NMR models
• Fit to SAXS data (no refine)

CCP10 CCP11 CCP12

• Fit looks good
• Use Fh10-12 model as single

rigid body for larger constructs SAXS envelope

• Shape envelopes determined from SAXS data alone
• DAMMIF (Franke & Svergun, 2009)

6.5 nm 7.1 nm

Fh 11-12

Ab initio modelling: Factor-H

Fh 12-13

Ab initio modelling: Factor-H

• Larger constructs

10.4 nm 10.5 nm 8.9 nm

Schmidt et al. (2010), J. Mol. Biol., 395:105-22.

Fh 10-15 Fh 11-14 Fh 10-12

Ab initio modelling: Factor-H

• The ab initio models provide:
• Indication of compaction for Fh10-15
• Compact end for Fh10-15?
• 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 of complexes

• Finds optimal configuration of subunits in a complex
• I(s) calculated using spherical harmonics
• Multiple curves, contrasts & symmetry supported
• User-defined contacts & orientation constraints
• Minimises steric clashes

Rigid body modelling

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
• SASREF
• BUNCH/CORAL (models linkers)
• Using overlapping NMR models
• Low temperature refinement
• Decrease possible solutions

Rigid body modelling: Fh11-14

• Using Fh11-12 and single CCPs
• CCP13 overlaid as in NMR

structure.

• CCP14 free to move
• Low temperature refinement

CCP14 CCP13 CCP12 CCP11

Rigid body modelling: Fh10-15

• CCPs 10-13 fixed
• Good fit but CLASHES
• Free CCP10
• Good fit no clashes
• Flexible 10-11 linker?
• NOT a horse-shoe shaped structure

10-13 fixed CCP10 free

CCP15 CCP13 CCP14 CCP12 CCP11 CCP10

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)
• 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)
• 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
• How does the program work?
• Generation of random pool of

models.

• Genetic algorithm.
• Best ensemble selected.
• Provides Rg & Dmax

distributions

Flexibility analysis by EOM

• eg. Poly-ubiquitin
• data (single structure)
• data (flexible case)
• Rg distributions
• Broad = flexible
• Narrow = "rigid"
• P. Bernado,(2010), Eur Biophys. J 39(5) 769-80

Flexibility: Factor-H

• Fh 12-13 extended, "rigid"
• Fh 11-14 compact, "rigid"
• Fh 10-15 compact, more flexible?

Mertens & Svergun, J Struct Biol. 2010 Oct;172(1):128-41

FactorH-C3b interaction

C3b

• Prepositioned for interaction
• Simultaneous recognition of multiple

binding sites Factor-H

FactorH-C3b

“By the way Haydyn, we just solved the FactorH-C3d crystal structure... can you do some more SAXS?”

• Paul Barlow (2010)

FactorH-C3d crystal structure

• 1:1 C3 – FactorH (19-20) complex
• SPR/NMR used to check interface
• Interface involves CCP19 and 19-20 linker
• C3d and C3b binding is equivalent
• aHUS mutations across interface

Morgan et al., Nat Struct Mol Biol. 2011 Apr;18(4):463-70.

FactorH-C3 interaction model

• Crystal
• Factor-H (19-20):C3d, (1-4):C3
• NMR
• FH 5, FH 6-8
• SAXS models
• FH 1-4, FH 8-15, FH 15-19

FH8-15 FH6-8 FH15-19 C3b C3d:FH19-20 Overlay: C3b:FH1-4 and C3d-FH19-20 SAXS FH1-4 and C3b:FH1-4

FactorH:C3 crystal structures comparison

• Both structures very similar
• Stoichiometry different

1:2 1:1 aligned with 1:2

Summary

• Modular systems well suited to combined SAXS/NMR

approaches

• Module structures solved by NMR
• Modules combined using SAXS
• Factor-H central core shown to be compact and

relatively rigid but maybe 10-11 flexible linker?

• Crystal structures suggest either 1:1 or 1:2 complex
• Data supports bent-back FH structure

– More constructs being analysed:

• Factor-H:C3b complex

• EMBL-Hamburg
• D.I. Svergun
• M.V. Petoukhov
• M.W. Roessle
• A.G. Kikhney
• D. Franke
• C. Blanchet
• M. Gadja
• C. Gorba
• P. Konarev
• W. Shang
• A. Shkumatau
• University of Edinburgh
• Paul Barlow & Jon Hannan
• Christoph Schmidt
• Eliza Markou

Acknowledgements