Combining SAXS and crystallography Rob Meijers EMBL Hamburg - - PowerPoint PPT Presentation

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

22/10/16 2

Haydyn Mertens

The before and after of Endolysins

• Endolysins lyse

bacteria

• 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

• Endolysins

Cd27l & CS74L: N-acetylmuramoyl-L-alanine amidase

• Ctp1l: Glycosyl hydrolase (lysozyme)

Courtesy Matthew Dunne

• C. Tyrobutyricum causes cheese blowing

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Gouda cheeses Blown

• CTP1L endolysin

works on harmless Clostridium

• Looks very much like

the endolysin that targets 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 confirms

difference in states between wild-type and C238R

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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 different

stochiometries of Netrin: DCC56

• Use OLIGOMER to

calculate summation

• f free,1:1 and 1:2

complexes

• SASDBD entry

SASDA76

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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 axon

guidance

• Heparan sulfate &

chondriate sulfate derivatives

• KO of enzymes

involved in production changes wiring of the brain

22/10/16 42

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