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NMR and SAXS: Two complementary techniques Annalisa Pastore NIMR - - PowerPoint PPT Presentation
NMR and SAXS: Two complementary techniques Annalisa Pastore NIMR - - PowerPoint PPT Presentation
NMR and SAXS: Two complementary techniques Annalisa Pastore NIMR A bit of NMR history Nuclear Magnetic Resonance 1 Gauss 23.5 Tesla Circa 500.000 times >> The magnetic field removes the degeneracy of the nuclear spin levels NMR as a
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Nuclear Magnetic Resonance
1 Gauss 23.5 Tesla Circa 500.000 times >>
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The magnetic field removes the degeneracy of the nuclear spin levels
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NMR as a radio…
Tune the frequency to observe a certain… element….
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The 1H spectrum of a protein
The position of each peak on the spectrum is called chemical shift
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A 13C spectrum
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We can mix the channels
Excite one nucleus, transfer the magnetization to another, and than back to the first
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You may gain resolution increasing the dimensions
Like a 2D Gel
The fingerprint
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Chemical shift perturbation as a tool to map interactions…
Sensitivity to the chemical environment
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It is used to study proteins because:
- Structure determination in solution
- Intermolecular interactions
- Dynamics
- Weak interactions
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The linewidth is roughly proportional to the tumbling time in solution
We are limited in the molecular size we can afford…
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A cut-and-paste approach
SAXS can help…
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NMR SAXS
A pact of friendship...
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The example of frataxin
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Friedreich’s ataxia
A lethal neurodegenerative disease
- Is the most frequent hereditary ataxia
- 1 in 50,000 affected individuals
but (being recessive)
- 1 in 120 carriers in European population!!!
Associated with: – Progressive gait and limb ataxia – Lack of leg reflexes – Disarthria – Diabetes mellitus
- Death often caused by cardio-hypertrophy
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The Frataxin iron binding site is only composed of Glu and Asp
Nair et al. (2004) Structure 12, 2037.
Fe titration of frataxin
A rather unsual coordination!
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What is frataxin function?
A thick fog…
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Increasing evidence links frataxin to Fe-S cluster assembly…
Bioinformatic evidence Huynen et al. (2001) Hum. Mol.
- Genet. 10, 2463-2468
Genetic and biochemical evidence Gerber et al. (2003) EMBO reports 9, 906 Layer et al. (2006) JBC 10,1074 Muhlenhoff et al. (2002) EMBO J. 22, 4815 Ramazzotti et al. (2004) FEBS Lett. 557, 215 Yoon and Cowan (2003) JACS 125, 6078
GST-pull down
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Iron sulfur clusters
Iron sulphur clusters are the eldest response to the problem of storing iron and sulfur in a non-toxic form
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2Cys 2Ala + S-S Converts Cys into Ala
Fe-S assembly is centred on a desulphurase and a transient acceptor
IscS dimer = desulphurase IscS iscU Cys Ala Fe2+ IscS iscU IscS iscU
+
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- CyaY in vivo interacts with the desulfurase IscS.
- IscU and CyaY bind to IscS but do not compete
GST GST-CyaY Pull-down
IscS
Protein interactions
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We have the structures of all three components…
What is the structure of the complex?
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Getting crystals of IscS/IscU or IscS/frataxin complexes
A different approach… based on NMR, SAXS and mutagenesis
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The pipeline…
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Frataxin and IscU do NOT compete for the same IscS surface
30 35 40 45 50 55 5 10 15
[IscS] (µM) Fluorescence
Kd (IscU) = 0.9 +/- 0.4 µM
30 32 34 36 38 40 42 44 46 48 50 5 10 15
[IscS] (µM) Fluorescence
Kd (IscU) = 1.2 +/- 2 µM
- frataxin
+ frataxin
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What are the consequences of these interactions?
IscS-IscU
[IscU] nM Kd=1.5 µM Kd=200 nM
0.15 0.3 0.45 100 200 300 400 500 600 CyaY noCyaY
Addition of Cyay to IscS increase IscU affinity
[IscS] μM CyaY- IscS Kd=20 µM
Kd=35 nM
0.5 1 1.5 2 2.5 3 10 20 30 40 50 60
noIscU IscU
Addition of IscU to IscS increase CyaY affinity
Synergic interaction!!!
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SAXS (Small-angle X-ray Scattering)
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SAXS to model the complexes
We characterized the binary and tertiary complexes This technique gives information on the shape and size of macromolecules The envelops of the complexes are different from the one of single species Identification of the excess volumes for monomeric IscU and CyaY
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How can we get the interaction surfaces?
IscS frataxin IscU
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The interacting surface maps
- nto a conserved region of
Frataxin
the residues involved are those necessary for iron binding!
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HADDOCK calculations
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Distance restraints
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OUR SAXS + NMR model…
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pdb: 3LVL
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The crystal structure in our model…
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The low resolution structure of the CyaY/IscS complex
R220 R223 R225
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The ternary IscS/IscU/CyaY complex
The interaction is iron independent
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Validating the model…
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Only the IscS_I314E_M315E mutant does not bind to IscU
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Only IscS_R220ER223ER225E mutant abolishes binding to frataxin
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The whole pipeline
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Saxs and NMR as complementary techniques! To model big multidomain proteins and complexes To validate a structure
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The Isc proteins using E. coli as a model system
Isc operon
IscR 2 IscS IscU IscA hscB hscA fdx YfhJ
- E. coli
Our ultimate aim is understanding the link between Fe/S formation and frataxin
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66 amino acid protein -> smaller than CyaY (106 residues) But, like frataxin, Highly acidic (PI=3.7) Binds to Iron Binds to IscS Competes with Frataxin on IscS
IscX:
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IscX binds IscS… in the same site as Frataxin
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Two binding sites on IscS?
1 2 3 4 6 5 7
75 50 37 25 100 150 250
1:0 1:1 1:20 1:40 1:0 IscS:IscX CL CL CL CL
- CL Cross-Linker
Maldi-TOF indicates both 1:1 52kDa and 60kDa IscS:IscX complexes Site 2 has a binding affinity <<< lower than site 1
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Can SAXS help also in this case?
K4 R220,223,225 K4 K101 K4 R220,223,225
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Problems…
When we have a good fit the models do not agree with cross-linking The best models in terms of cross-linking have a poor fitting Can we do any better?
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Acknowledgements
Filippo Prischi, Salvatore Adinolfi, Clara Iannuzzi, Robert Yan, Rita Puglisi Peter Konarev and Dmitri Svergun (EMBL)