combining saxs and crystallography - embl … · acknowledgements • embl hamburg • matthew...
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Combining SAXS and crystallography
Rob Meijers EMBL Hamburg Outstation, Germany
Stories of SAXS and MX
• A dimer that didn’t add up
• A trimer that didn’t add up
• A disordered guidance cue
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Haydyn Mertens
The before and after of Endolysins
• Endolysins lysebacteria
• From the inside• And the outside
Rapid lysis of Listeria monocytogenes cells by exogenous addition of endolysin (Loessner 2005)
C. difficile persistence • C. difficile is an anaerobic Gram-
positive bacterium• Spores resistant to heat,
detergents• Strains are resistant to most
antibiotics, including vancomycin
http://www.msnbc.msn.com/id/24407803/wid/17621070; Dr. Curtis Donskey, Clinical Infectious Diseases, February 2008.
• Colonizes gut after antibiotic treatment• Causes colitis
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Endolysins
Cd27l & CS74L: N-acetylmuramoyl-L-alanine amidase
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Ctp1l: Glycosyl hydrolase (lysozyme)
Courtesy Matthew Dunne
C. Tyrobutyricum causes cheese blowing
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Gouda cheeses
Blown
• CTP1L endolysinworks on harmlessClostridium
• Looks very much likethe endolysin thattargets C. Diff
Clostridia contamination
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Put this endolysin into Lacto-coccus
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Lacto-coccus
Clostridium
Why doesn’t it work?
• Need both catalytic and cell wall binding domain to lyse bacteria
• When introduced in L. Lactis, it is not efficient
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Activation of endolysin
Korndoerfer et al. (2006) J. Mol. Biol. 364, 678
PlyC endolysin
8 x Cell wall binding domains
One catalytic domain Gowan et al (2012) PNAS 109, 12752
CD27L mutational studies
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buffer
C239S
wildtype
Removing disulfide reduces lytic activity. Periplasm is an oxidising environment perfect for disulfide formation
Removing disulfide bond in CD27L reduces catalytic activity
Dunne et al. Plos Path (2014) 7, 104228
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)
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C238R is a pure dimer
• OLIGOMER confirmsdifference in statesbetween wild-type andC238R
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Complex between full length and truncated endolysin • Where does the
truncated C-terminal domain come from?
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Matthew Dunne
So what does the mix consist of??
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Confirmed by native mass spectrometry
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CTP1L
Confirmed by native mass spectrometry
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CD27L: a bigger mess
The extra bit plays a role in activity
A Shine-Dalgarno sequence
CTP1L/CS74L
CD27L
Dunne et al. (2016) JBC 291(10):4882-93
An internal translation site
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Lys-Gly AAG GGG
Val GTG
Silent mutation: AAG GCG
RBS knockout: Ser-Ser
Put the genetic switch into Lacto-coccus
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Lacto-coccus
Clostridium
A trimer that didn’t add up
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Crossing the midline
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Mirror movements
Srour et al. Science (2010) 328, 592
Neural tube
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Chetodal Trends in Neurosciences (2014) 37, 551
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Getting around
Hunger
Course
Alarm clock
Scary physicists
Chemo-attraction and repulsion towards netrin
Attraction Repulsion
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DCC receptor DCC + UNC5A receptor
Courtesy Paul Letourneau
Netrin as a guidance cue
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Netrin-soaked bead
Neuron
Axon
Attraction
DCC + UNC5
Repulsion
Netrin DCC
DCC, UNC5 and DSCAM are Netrin receptors
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UNC5
DCC
DSCAM
A large signaling complex is formed
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Ko et al Trends in Molecular Medicine (2012) 8, 9
Getting netrin and DCC together
Nina Krüger
Human Netrin-1 Lorenzo Finci (Beijing University)
Fn456
Strep tag
DCC receptor
The individual molecules are monomeric
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DCC fit in ab initio bead model
Courtesy Haydyn Mertens
Netrin with extended glycosylation
A closer look
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Finci et al (2014) Neuron 83, 839
Structure of netrin-1/DCC fragment
DCC
Cluster
DCC
Chemo-attraction complex
DCC
Cluster
DCC
Site1: Hotspot
Netrin/DCC binding Axon guidance
A unique protein/protein binding site
DCC
Cluster
DCC
Sulfate ions Chloride ion
Correlation between binding and chemo-attraction
DCC
Cluster
DCC
DCC/Netrin binding
Axon guidance (chemo-attraction)
Can we ‘see’ the 2 sites in solution?
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DCC
Cluster
DCC
Using SAXS to study binding kinetics
• Mix differentstochiometries ofNetrin: DCC56
• Use OLIGOMER tocalculate summationof free,1:1 and 1:2complexes
• SASDBD entrySASDA76
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Site 1 DCC mutant behaves differently
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DCC
Cluster
DCC M933R
A complementary structure
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Composite model with Xu et al Science (2014) 344, 1275
Netrin
DCC
DCC
Heparin binding site?
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Sulfate ions
The third component in guidance?
• Sugar code in axonguidance
• Heparan sulfate &chondriate sulfatederivatives
• KO of enzymesinvolved in productionchanges wiring of thebrain
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Hypothesis: small molecules act as receptor selectors
Acknowledgements
• EMBL Hamburg• Matthew Dunne• Nina Krueger• Tuhin Bhowmick• Xuefan Gao• Haydyn Mertens• Dmitri Svergun
• EMBL Heidelberg• Stefan Leicht• Jeroen Krijgsveld
• HPI Hamburg• Charlotte Utrecht• Boris Krichel
• Norwich IFR• Melinda Mayer• Arjan Narbad
• Soleil• Andrew Thompson
• Peking University• Ying Liu• Yiqiong Liu• Yu Wu• Yan Zhang• Junyu Xiao• Lorenzo Finci
• DFCI• Jia-huai Wang