Joint use of SAXS and crystallography Rob Meijers EMBL Hamburg - - PowerPoint PPT Presentation

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Joint use of SAXS and crystallography Rob Meijers EMBL Hamburg - - PowerPoint PPT Presentation

Jul 30, 2023 128 likes •681 views

Joint use of SAXS and crystallography Rob Meijers EMBL Hamburg Outstation SAXS EMBO Course EMBL Hamburg 2018 Stories of SAXS and MX Following lipid mediated protein complex formation SAXS as the arbiter for cell surface receptor

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Joint use of SAXS and crystallography

Rob Meijers EMBL Hamburg Outstation SAXS EMBO Course EMBL Hamburg 2018

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Stories of SAXS and MX

  • Following lipid mediated protein

complex formation

  • SAXS as the arbiter for cell surface

receptor dimerization

  • A disordered guidance cue

23/11/18 2

Haydyn Mertens

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SLIDE 3

Crystal structure 1:1 SAXS

  • SAXS user1: unfolded
  • SAXS user2: globular

dimer

  • Call from above: a rod

24/11/18 3

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Example1: The crystal structure just doesn’t fit

24/11/18 4

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Endolysins

24/11/18 5

  • Cd27l & CS74L: N-acetylmuramoyl-L-alanine amidase
  • Ctp1l: Glycosyl hydrolase (lysozyme)
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Crystal structure is confusing

24/11/18 6

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So what does the mix consist of??

24/11/18 7

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SAXS + mutagenesis clears up oligomeric state

  • Wild-type CD27L does

not fit a dimer, nor a tetramer (SASDAL5)

  • But C238R mutant fits

a dimer (SASDAM5)

24/11/18 8

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C238R is a pure dimer

  • OLIGOMER confirms

difference in states between wild-type and C238R

24/11/18 9

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A Shine-Dalgarno sequence

CTP1L/CS74L CD27L

Dunne et al. (2016) JBC 291(10):4882-93

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SLIDE 11

24/11/18 11

An internal translation site

Lys-Gly

AAG GGG

Val

GTG

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Titrating a weak complex

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The dark side of DCC

23/11/18 13

Mehlen et al, Nature Reviews in Cancer 2011, 11, 188

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Structure of netrin/DCC fragment

23/11/18 14

Finci et al (2014) Neuron 83, 839

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Using SAXS to study binding kinetics

  • Mix different

stochiometries of Netrin: DCC56

  • Use OLIGOMER to

calculate summation

  • f free,1:1 and 1:2

complexes

  • SASDBD entry

SASDA76

23/11/18 15

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Site 1 DCC mutant behaves differently

23/11/18 16

DCC Cluster DCC M933R

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Analyzing a partially folded protein

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Draxin binds to DCC receptor

  • Deleted in colorectal

cancer

  • Netrin & draxin binding

to different regions

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Axon -1 Netrin Draxin SP DCC Netrin Draxin axin

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Draxin is partially unfolded

Draxin-C Draxin-22

Liu et al. Neuron 2018

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The "unfolded-ness" or "random coil" likeness of a biological macromolecule can be qualitatively assessed by means of a Kratky plot. The small angle X-ray scattering (SAXS) data of human full length Draxin clearly show the protein is partially unfolded in solution.

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EOM ¡shows ¡another ¡aspect ¡

  • Wide ¡distribu5on ¡of ¡Rg ¡
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Two adaptor proteins that can only form a complex when PIP2 lipid is present

23/11/18 22

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Clathrin-mediated Receptor endocytosis

Credit: Journal of Cell Science

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Clathrin-mediated endocytosis

Lauwers et al. (2016) Neuron 90, 11

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Phosphorylation rules adaptor protein binding

Schill & Anderson Biochem. J. 2009 418, 247-260

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Two adaptor proteins that work as connectors

  • Adaptor proteins connect

clathrin to cell membrane

  • Ent1 and Sla2 connect cell

membrane to actin cytoskeleton

  • Some ANTH connect to

AP2 (which binds cargo)

Wood & Royle Dev Cell 2015

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Different assemblies between Human and fungal CME

Merrifield & Kaksonen (2014), CSH Perspec. Biol. 6:a016733

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Mechanisms for curving the membane

Merrifield & Kaksonen (2014), CSH Perspec. Biol. 6:a016733

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ENTH and ANTH bind through PIP2

24/11/18 29

Skruzny et al. Developmental Cell, 2015

ENTH ANTH PIP2

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Human epsin oligomers observed with EPR

Yoon et al. (2010) JBC 285, 531

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ITC shows a 1:1 complex for ENTH:ANTH

Skruzny et al. Developmental Cell, 2015

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EM shows large GUV vesicles with ENTH and ANTH embedded

Skruzny et al. Developmental Cell, 2015

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Fitting the complex in the blobs

Skruzny et al. Developmental Cell, 2015

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Let’s look at the assembly of the complex

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yENTH2 forms a PIP2-associated dimer

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Compared to IP3- structure, N-terminal helix is oriented differently

  • IP3 lacks the aliphatic

tail

  • We cannot see the tail

either except for the 1st carbon

  • Orientation of lipid is

different also

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SLIDE 37

This makes space for a second ENTH module to bind

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Ready to form larger oligomers

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Mass spectrometry

  • MS/MS + Ion mobility :
  • Detailed folding state
  • Protein-protein

interactions

  • Whole protein size…

Courtesy Waters

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Native mass spec used to look at stoichiometry

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yENTH/PIP2 needs Sla2 to form larger complex

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yENTH/Sla2/PIP2 complex matures in 3 minutes

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But human assembly is different

ENTH hexamer alone

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How does human epsin/PIP2 look like?

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Small angle X-ray scattering on human ENTH1

  • Created “filled” dimer
  • f yENTH2
  • Used 3 copies to fill

hexamer

  • Applied P3 symmetry
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SAXS dummy model vs construct

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And the structure, in solution?

Side view Membrane view

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Model for epsin/Sla2 assembly in yeast

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Model for epsin/Hip1R assembly in human

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Mechanisms for curving the membane

Merrifield & Kaksonen (2014), CSH Perspec. Biol. 6:a016733

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You can mix epsins, complex still forms

  • Mixing ENTH1 and

ENTH2 gives complex with 4 ENTH1 and 4 ENTH2 molecules

  • Despite 73 %

sequence identity

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You can mix species, complex still forms…

cENTH/ySLA2 hENTH/ySla2 or hENTH/ cSla2 ySLA2 cSLA2

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Stochastic assembly of adaptors

  • PIP2-driven
  • ligomerization
  • Adaptor recruitment
  • Is stochastic
  • Depends on available

adaptors

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Acknowledgements

  • EMBL Hamburg
  • Maria Garcia-Alai
  • Nina Krueger
  • Matt Dunne
  • Tuhin Bhowmick
  • Sandra Kozak
  • Ioana-Maria Nemtanu
  • Stephane Boivin
  • Haydyn Mertens
  • Dmitri Svergun
  • Gleb Bourenkov
  • Thomas Schneider
  • Peking University
  • Yiqiong “Helen” Liu
  • Junyu Xiao
  • Ying Liu
  • Yan Zhang
  • Max Planck Marburg
  • Michael Skruzny
  • Dana Farber Cancer Institute
  • Jia-huai Wang
  • University of Geneva
  • Marko Kaksonen

Heinrich Pette Institute

  • Johannes Heidemann
  • Charlotte Uetrecht