Two complementary techniques Annalisa Pastore NIMR We need both - - PowerPoint PPT Presentation

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Two complementary techniques Annalisa Pastore NIMR We need both - - PowerPoint PPT Presentation

Feb 04, 2024 323 likes •970 views

Joint use of NMR and SAXS: Two complementary techniques Annalisa Pastore NIMR We need both Details Full picture The main techniques to solve structure X-ray crystallography Electron Microscopy Nuclear Magnetic Resonance Today number of

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Joint use of NMR and SAXS: Two complementary techniques

Annalisa Pastore NIMR

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We need both…

Details Full picture

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The main techniques to solve structure

Nuclear Magnetic Resonance X-ray crystallography Electron Microscopy

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Today number of structures in PDB

02/05/13 90206 structures NMR EM 538 9905 X-ray 79,541

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We all have limits…

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The limits of X-ray…

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The linewidth is roughly proportional to the correlation time

We are limited in the molecular size we can afford…

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We think of proteins as globular

  • bjects…

Instead….

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…combinatorial possibilities

A limited number of building blocks is sufficient to produce different proteins...

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Proteins are like tangrams…

A limited number of building blocks is sufficient to produce different figures

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A cut-and-paste approach

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Cut-and-paste…

Solve the structures of individual parts… …and riconstruct the overall picture by SAXS

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Some examples

  • Titin: how to obtain the overall shape
  • Ataxin 3: how to validate a structure
  • High molecular weight complexes
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The Sarcomere

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How can we solve the structure

  • f titin?
  • It is 3 Million Da (ca. 27,000 residues)
  • It is a filament ca. 1 m long
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Selection of three modules as:

A71

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What is their relative orientation?

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Titin modules are assembled as beads in a necklace

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Bases of passive elasticity

Von Castelmur et al., PNAS 2007

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The case of ataxin-3

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It leads to great motor disability, without ever altering the intellect, until the death of the patient.

Spinocerebellar ataxia type-3 (SCA3) or Joseph-Machado disease (MJD) is hereditary and dominant

Thought to have originated from founders in the Iberia Peninsula, who migrated to the Azores and to other countries including Japan It is caused by polyQ expansion in ataxin 3, a small protein (42 kDa) mapped on chromosome 14 (14q32.1)

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PolyQ diseases are dominant hereditary pathologies caused by a trinucleotide expansion

CAG Expansion in coding regions (CAG)n (Gln)n n<35 folded protein n>35 Toxic aggregates

At least 8 Polyglutamine (polyQ) expansion diseases

Apoptosis

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The solution structure of ataxin-3

…consists of the Josephin domain and of an unstructured tail which comprises polyQ

Masino et al. (2002) Febs Lett. 513, 267-272 Josephin

polyQ

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GST-Ub52 Glu9 Cys14 His119 Asn134

Josephin is a polyUb cysteine protease

Josephin 1-182

polyQ

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The hairpin behaves as a waving hand

MD simulations

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One month later…

Which is the correct structure?

2aga 1yzb

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Small angle scattering

Only one structure fits the data: ours!!!

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Structure of the josephin/Ubiquitin complex

Complex josephin/ distal Ub

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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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and partial silencing of frataxin

Friedreich’s ataxia is caused by expansion of the trinucleotide GAA

Campuzzano et al., 1996

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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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Frataxin binds IscS weakly but stoichiometrically

Kd 27+/- 2 uM N 0.97 +/- 0.05 Calorimetry studies

We pull-down endogenous IscS IscS is bound to IscU

Frataxin

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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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SAXS (Small-angle X-ray Scattering)

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SAXS on IscS

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SAXS DATA on IscS+IscU IscS – IscU

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SAXS of IscS+frataxin

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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 CyaY

the residues involved are those necessary for iron binding!

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180° IscU

Interacting surface on IscU

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HADDOCK calculations

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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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Search for a complementary patch of residues on IscS

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Only IscS_R220ER223ER225E mutant abolishes binding to frataxin

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We are now using this approach…

110 kDa complexes and more…

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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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Acknowledgements

Titin project: Sabina Improta, Mark Pfuhl, Claudia Muhle, Catherine Joseph Siegfried Labeit (EMBL), Mathias Gautel (EMBL), Jill Trewhella (Los Alamos) Ataxin-3 project: Giuseppe Nicastro, Laura Masino Dmitri Svergun (EMBL) Frataxin project: Filippo Prischi, Salvatore Adinolfi, Clara Iannuzzi, Peter Konarev and Dmitri Svergun (EMBL)

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Experimental Hutch

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Nuclear Overhauser Effect (NOE) for protons at distance not > 5 A

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Distance restraints

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NOESY

2D NOE map (NOESY): a distance map of the molecule

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A distance map of the protein

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NMR SAXS

A pact of friendship...

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We need both…