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 Haydyn Mertens 2 22/10/16

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 � ������������ ������������� ������ ������� ��������� ��� ��� ��� ��� Ctp1l: Glycosyl hydrolase (lysozyme) Cd27l & CS74L: N-acetylmuramoyl-L-alanine amidase Courtesy Matthew Dunne

C. Tyrobutyricum causes cheese blowing • CTP1L endolysin Gouda cheeses works on harmless Clostridium • Looks very much like the endolysin that targets C. Diff Blown 6 21/10/16

Clostridia contamination 7 21/10/16

Put this endolysin into Lacto-coccus Lacto-coccus Clostridium 8 21/10/16

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 9 21/10/16

Activation of endolysin PlyC endolysin 8 x Cell wall binding domains One catalytic domain Gowan et al (2012) PNAS 109, 12752 Korndoerfer et al. (2006) J. Mol. Biol. 364 , 678

CD27L mutational studies Removing disulfide bond in CD27L reduces catalytic activity buffer C239S Removing disulfide reduces lytic activity. Periplasm is an oxidising wildtype environment perfect for disulfide formation Dunne et al. Plos Path (2014) 7, 104228 11 21/10/16

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) 12 22/10/16

C238R is a pure dimer • OLIGOMER confirms difference in states between wild-type and C238R 13 21/10/16

Complex between full length and truncated endolysin • Where does the truncated C-terminal domain come from? Matthew Dunne 14 21/10/16

So what does the mix consist of?? 15 21/10/16

Confirmed by native mass spectrometry CTP1L 16 21/10/16

Confirmed by native mass spectrometry CD27L: a bigger mess 17 21/10/16

The extra bit plays a role in activity

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 Silent mutation: AAG GCG RBS knockout: Ser-Ser 20 21/10/16

Put the genetic switch into Lacto-coccus Lacto-coccus Clostridium 21 21/10/16

A trimer that didn’t add up 22 21/10/16

Crossing the midline Mirror movements Srour et al. Science (2010) 328 , 592 23 21/10/16

Neural tube Chetodal Trends in Neurosciences (2014) 37 , 551 24 21/10/16

Getting around Course Hunger Scary physicists Alarm clock 25 22/10/16

Chemo-attraction and repulsion towards netrin Repulsion Attraction DCC receptor DCC + UNC5A receptor Courtesy Paul Letourneau 26 21/10/16

Netrin as a guidance cue Attraction Repulsion Neuron DCC Axon Netrin Netrin-soaked bead DCC + UNC5 27 21/10/16

DCC, UNC5 and DSCAM are Netrin receptors DSCAM DCC UNC5 28 22/10/16

A large signaling complex is formed Ko et al Trends in Molecular Medicine (2012) 8 , 9 29 22/10/16

Getting netrin and DCC together DCC receptor Nina Krüger Fn456 Strep tag Lorenzo Finci (Beijing University) Human Netrin-1

The individual molecules are monomeric DCC fit in ab initio bead model Netrin with extended glycosylation Courtesy Haydyn Mertens 31 21/10/16

A closer look Finci et al (2014) Neuron 83, 839 32 21/10/16

Structure of netrin-1/DCC fragment DCC DCC Cluster

Chemo-attraction complex DCC DCC Cluster Site1: Hotspot Axon guidance Netrin/DCC binding

A unique protein/protein binding site DCC DCC Cluster Chloride ion Sulfate ions

Correlation between binding and chemo-attraction DCC/Netrin binding DCC DCC Axon guidance (chemo-attraction) Cluster

Can we ‘see’ the 2 sites in solution? DCC DCC Cluster 37 21/10/16

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 38 21/10/16

Site 1 DCC mutant behaves differently M933R DCC DCC Cluster 39 21/10/16

A complementary structure Netrin DCC DCC Composite model with Xu et al Science ( 2014) 344, 1275 40 22/10/16

Heparin binding site? Sulfate ions 41 22/10/16

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 42 22/10/16

Hypothesis: small molecules act as receptor selectors

Acknowledgements • Norwich IFR • EMBL Hamburg • Peking University Melinda Mayer • Matthew Dunne • • Ying Liu Arjan Narbad • Nina Krueger • • Yiqiong Liu • Soleil Tuhin Bhowmick • Andrew Thompson • Yu Wu • Xuefan Gao • Haydyn Mertens • • Yan Zhang Dmitri Svergun • • Junyu Xiao • EMBL Heidelberg • Lorenzo Finci Stefan Leicht • • DFCI Jeroen Krijgsveld • • Jia-huai Wang • HPI Hamburg Charlotte Utrecht • Boris Krichel •