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 dimerization • A disordered guidance cue Haydyn Mertens 2 23/11/18
Crystal structure 1:1 SAXS • SAXS user1: unfolded • SAXS user2: globular dimer • Call from above: a rod 3 24/11/18
Example1: The crystal structure just doesn’t fit 4 24/11/18
Endolysins � ������������ ������������� ������ ������� ��� ��� ��������� ��� ��� Ctp1l: Glycosyl hydrolase (lysozyme) Cd27l & CS74L: N-acetylmuramoyl-L-alanine amidase 5 24/11/18
Crystal structure is confusing 6 24/11/18
So what does the mix consist of?? 7 24/11/18
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) 8 24/11/18
C238R is a pure dimer • OLIGOMER confirms difference in states between wild-type and C238R 9 24/11/18
A Shine-Dalgarno sequence CD27L CTP1L/CS74L Dunne et al. (2016) JBC 291(10):4882-93
An internal translation site Lys-Gly Val AAG GGG GTG 11 24/11/18
Titrating a weak complex
The dark side of DCC Mehlen et al, Nature Reviews in Cancer 2011, 11, 188 13 23/11/18
Structure of netrin/DCC fragment Finci et al (2014) Neuron 83, 839 14 23/11/18
Using SAXS to study binding kinetics • Mix different stochiometries of Netrin: DCC56 • Use OLIGOMER to calculate summation of free,1:1 and 1:2 complexes • SASDBD entry SASDA76 15 23/11/18
Site 1 DCC mutant behaves differently M933R DCC DCC Cluster 16 23/11/18
Analyzing a partially folded protein
Draxin binds to DCC receptor • Deleted in colorectal Axon -1 cancer • Netrin & draxin binding Netrin to different regions Draxin axin DCC Draxin SP Netrin 18
Draxin is partially unfolded Draxin-22 Draxin-C Liu et al. Neuron 2018
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 .
EOM ¡shows ¡another ¡aspect ¡ • Wide ¡distribu5on ¡of ¡Rg ¡
Two adaptor proteins that can only form a complex when PIP2 lipid is present 22 23/11/18
Clathrin-mediated Receptor endocytosis Credit: Journal of Cell Science
Clathrin-mediated endocytosis Lauwers et al. (2016) Neuron 90, 11
Phosphorylation rules adaptor protein binding Schill & Anderson Biochem. J. 2009 418, 247-260
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
Different assemblies between Human and fungal CME Merrifield & Kaksonen (2014), CSH Perspec. Biol. 6:a016733
Mechanisms for curving the membane Merrifield & Kaksonen (2014), CSH Perspec. Biol. 6:a016733
ENTH and ANTH bind through PIP2 PIP2 ENTH ANTH Skruzny et al. Developmental Cell, 2015 29 24/11/18
Human epsin oligomers observed with EPR Yoon et al. (2010) JBC 285, 531
ITC shows a 1:1 complex for ENTH:ANTH Skruzny et al. Developmental Cell, 2015
EM shows large GUV vesicles with ENTH and ANTH embedded Skruzny et al. Developmental Cell, 2015
Fitting the complex in the blobs Skruzny et al. Developmental Cell, 2015
Let’s look at the assembly of the complex
yENTH2 forms a PIP2-associated dimer
Compared to IP3- structure, N-terminal helix is oriented differently • IP3 lacks the aliphatic tail • We cannot see the tail either except for the 1 st carbon • Orientation of lipid is different also
This makes space for a second ENTH module to bind
Ready to form larger oligomers
Mass spectrometry • MS/MS + Ion mobility : • Detailed folding state • Protein-protein interactions • Whole protein size … Courtesy Waters
Native mass spec used to look at stoichiometry
yENTH/PIP2 needs Sla2 to form larger complex
yENTH/Sla2/PIP2 complex matures in 3 minutes
But human assembly is different ENTH hexamer alone
How does human epsin/PIP2 look like?
Small angle X-ray scattering on human ENTH1 • Created “filled” dimer of yENTH2 • Used 3 copies to fill hexamer • Applied P3 symmetry
SAXS dummy model vs construct
And the structure, in solution? Side view Membrane view
Model for epsin/Sla2 assembly in yeast
Model for epsin/Hip1R assembly in human
Mechanisms for curving the membane Merrifield & Kaksonen (2014), CSH Perspec. Biol. 6:a016733
You can mix epsins, complex still forms • Mixing ENTH1 and ENTH2 gives complex with 4 ENTH1 and 4 ENTH2 molecules • Despite 73 % sequence identity
You can mix species, complex still forms … hENTH/ySla2 or hENTH/ cENTH/ySLA2 cSla2 ySLA2 cSLA2
Stochastic assembly of adaptors • PIP2-driven oligomerization • Adaptor recruitment • Is stochastic • Depends on available adaptors
Acknowledgements • EMBL Hamburg • Dana Farber Cancer Institute Maria Garcia-Alai • Jia-huai Wang • Nina Krueger • • University of Geneva Matt Dunne • Tuhin Bhowmick • Marko Kaksonen • Sandra Kozak • Ioana-Maria Nemtanu • • Peking University Stephane Boivin • Yiqiong “Helen” Liu Haydyn Mertens • • Dmitri Svergun • Junyu Xiao • Gleb Bourenkov • Ying Liu • Thomas Schneider • Yan Zhang • • Max Planck Marburg Heinrich Pette Institute Michael Skruzny • • Johannes Heidemann • Charlotte Uetrecht
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