Insights into the mechanism of botulinum neurotoxin (BoNT) receptor binding and substrate cleavage from a structural perspective

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1 Insights into the mechanism of botulinum neurotoxin (BoNT) receptor binding and substrate cleavage from a structural perspective Axel Brunger Stanford University Conference on New Frontiers in Microbiology and Infection

2 Outline Ca 2+ triggered neurotransmitter release BoNT/B receptor binding BoNT/A substrate binding BoNT/A inhibitor development

3 Outline Ca 2+ triggered neurotransmitter release BoNT/B receptor binding BoNT/A substrate binding BoNT/A inhibitor development

4 Neuromuscular junction J. Heuser

5 SNARE conformational cycle during Ca 2+ triggered neurotransmitter release Jahn, Scheller, Nat. Rev. Mol. Cell, 2006

6 synaptic vesicle SNAREs are the core of the fusion machine trans cis plasma membrane synaptobrevin syntaxin SNAP-25 C N pre-fusion post-fusion

7 The SNARE core complex is conserved from yeast to man Yeast vs. Neuronal Yeast vs. Early Endosomal Yeast vs. Late Endosomal Sutton, Fasshauer, Jahn, Brunger, Nature, 1998 Antonin, Fasshauer, Becker, Jahn, Schneider, Nat.Struct.Biol, 2002 Ernst, Brunger, J.Biol.Chem., 2003 Zwilling, Cypoinka, Pohl, Fasshauer, Walla, Wahl, Jahn, EMBO J., 2007 Strop, Kaiser, Vrljic, Brunger, J.Biol.Chem., 2008

8 Conserved structural transitions during assembly Unfolded structured unstructured Partially folded Fully folded Fasshauer, Bruns, Shen, Jahn, Brunger, J.Biol.Chem.,1997 Fasshauer, Otto, Eliason, Jahn, Brunger, J.Biol.Chem., 1997 Rice, Brennwald, Brunger, FEBS Lett., 1997 Fiebig, Rice, Pollock, Brunger, Nature Struct Biol., 1999 Hazzard, Sudhof, Rizo, J. Biomol. NMR, 1999 Sorensen, Fasshauer, et al., EMBO J., 2006 Weninger, Bowen, Chu, Brunger, Structure, 2008

9 Botulinum neurotoxin Cell surface binding (heavy chain) Receptor-mediated endocytosis Endosomal translocation releases light chain protease SNARE proteolysis by light chain protease

10 Modular architecture of BoNT/A holotoxin Lacy, Stevens et al., Nat. Struct. Biol. 5, 898- (1998)

11 translocation receptor binding proteolysis HCC ] HC HCN HN LC Molecular basis for receptor and substrate recognition?

12 Outline Ca 2+ triggered neurotransmitter release BoNT/B receptor binding BoNT/A substrate binding BoNT/A inhibitor development

13 BoNT Receptor Recognition dual receptors: ganglioside (glycosphingolipids) and protein (Montecucco, Trends Biochem Sci. 1986). protein receptors are luminal domains of synaptic vesicle proteins: synaptotagmin II for BoNT/B (Nishiki, Kozaki et al., J.Biol.Chem. 1994; Dong, Chapman et al., JCB, 2003) synaptotagmins I and II for BoNT/G (Rummel, Binz et al., JBC, 2004) SV2C for BoNT/A (Dong, Chapman et al., Science, 2006) SV2A and SV2B for BoNT/E (Dong, Chapman et al., Mol. Biol. Cell, 2008)

14 Crystal structure of the BoNT/B HCC - synaptotagmin II complex 44 luminal domain of synaptotagmin II 60 receptor binding domain (HcB) of BoNT/B Rongsheng Jin, Nature, 2006

15 Mostly hydrophobic interactions

16 High affinity of BoNT/B - synaptotagmin II interaction ITC N = 1.1 Kd = 34 nm ΔH = -6.9 kcal mol -1 ΔS = 10.4 cal mol -1 K -1 ΔCp = -326 cal mol -1 K -1

17 Synaptotagmin mutations disrupt interaction Pull-down with GST-Syt-II binding of WT HCC wild-type F47A L50A F54A F55A E57K

18 BoNT/B HCC mutations disrupt interaction Pull-down with WT GST-Syt-II binding of mutant HCC wild-type V1118D K1192E F1194A A1196K F1204A

19 Single site BoNT/B HCC mutations disrupt toxicity Toxicity wild-type V1118D K1192E F1194A A1196K F1204A HcB mutations T. Binz, A. Rummel

20 BoNT/B ganglioside receptor binding site Mutations of ganglioside binding site disrupt interaction and toxicity (Rummel, Binz et al, PNAS 10, , 2007) Co-crystal structure of BoNT/B with sialactose (Swaminathan, Eswaramoorthy, et al., NSMB, 7, 693, 2000)

21 Dual receptor recognition

22 BoNT/B HCC - synaptotagmin II interaction High affinity and specificity Independent ganglioside and protein binding sites Interactions may set the stage for translocation

23 Outline Ca 2+ triggered neurotransmitter release BoNT/B receptor binding BoNT/A substrate binding BoNT/A inhibitor development

24 translocation receptor binding proteolysis LC

25 SNAREs are the targets of LC proteases BoNT/G BoNT/D BoNT/C C N synaptobrevin BoNT/B & TeNT BoNT/F N N C BoNT/C BoNT/A BoNT/E C syntaxin SNAP-25

26 Conserved core of all BoNT and TeNT LC proteases Recognition of the distinct targets?

27 Crystal structure of the BoNT/A LC protease SNAP-25[ ] complex N α exosite Mark Breidenbach, Nature 2004

28 Crystal structure of the BoNT/A LC protease SNAP-25[ ] complex N α exosite extended

29 Crystal structure of the BoNT/A LC protease SNAP-25[ ] complex N α exosite extended cut β exosite C

30 Structural transition of SNAP-25 upon binding assembled SNARE complex protects SNAP-25 from proteolysis

31 Mutants of interacting residues reduce catalytic efficiency K m k cat k cat /K m N N-terminal SNAP-25 truncations result in 55-fold increased K m with limited effect on k cat (Li, Binz,Niemann, Singh et al., Biochem 39, 2399-, 2000) C-terminal SNAP-25 truncations (beyond M202) cannot be cut by BoNT/A (Schmidt, Bostian, J. Prot. Chem 14, 703, 1995) Mutational studies are in agreement with crystal structure (Chen, Barbieri, J. Biol. Chem. 281, , 2006; Chen, Kim, Barbieri, J.Biol.Chem. 282, 9621-, 2007) M202 C S187 I156 M167

32 The extensive interface of the BoNT/A LC - SNAP-25 complex explains the high substrate specificity of the LC protease consistent with single-site and truncation mutagenesis studies BoNT/A LC captures SNAP-25 in a partially unstructured state

33 Outline Ca 2+ triggered neurotransmitter release BoNT/B receptor binding BoNT/A substrate binding BoNT/A inhibitor development

34 Inhibition of BoNT/A LC protease with peptidomimetics Inhibitor Inhibitor Sequence Ki (μm) Std. Dev. P1 P1 P2 P3 P4 P5 P6 SNAP-25 Q R A T K M L I2 3-Phenylpropanoyl- R A T K M L >200 I3 N-Acetyl-F R A T K M L >200 I4 [D]F R A T K M L >200 I5 F R A T K M L I6 G R A T K M L I7 DNP-DAB R A T K M L I8 DNP-DAB R BZA T K M L I9 DNP-DAB R BZA T DAB M L I10 DNP-DAB R F T K M L I11 DNP-DAB R W T ORN M L I12 DNP-DAB R W T DAP M L p435 DNP-DAB R W T DAB M L All peptides have a free amino group at the N-terminus unless noted otherwise, and the C-terminus is amidated. Non-standard abbreviations: DNP-DAB, 4-(2,4-dinitrophenylamino)-2-amino-butanoic acid; ORN, ornithine; DAB, 2,4-diaminobutanoic acid; DAP, 2,3-diaminopropanoic acid; BZA, benzothien-3-yl-alanine. All amino acids are the [L] stereoisomer unless noted otherwise. J. Schmidt et al.

35 Crystal structure of the complex of between BoNT/A LC and p435 Zn 2+ N 250s C 370s 60s p435 Ki = 41 nm SNAP-25 Jorge Zuniga, Structure, 2008

36 The inhibitor p435 accomplishes high affinity by binding in a very different conformation than substrate (SNAP-25) C L M DAB T W R DAB-DNP p435 Zn 2+ N. L M K T A R Q..... SNAP-25 Zn 2+ bound conformations from corresponding complexes with BoNT/A LC

37 Tight binding is accomplished by induction of new pockets not present in apo BoNT/A structures BoNT/A LC - p435 vs. apo BoNT/A LC DNP-DAB Trp Met Arg Leu Thr DAB

38 Induction of new pockets is key for high affinity p435 inhibitor Ki =41 nm

39 p435 inhibitor Ki =41 nm SNAP-25 substrate

40 p435 inhibitor Ki =41 nm N-Ac-CRATKML Ki = 1.9 μm (Silvaggi, Allen, et al. Biochemistry, 2008)

41 p435 inhibitor Ki =41 nm QRATKM Ki = 6 μm (Kumaran, Swaminathan, et al., PLOS Pathogens, 2008)

42 p435 inhibitor Ki =41 nm L-arg hydroxamate Ki = 50 μm (Silvaggi, Allen, et al. Chemistry & Biology, 2007)

43 p435 is a highly specific inhibitor (tested at 4 μm) Protease Substrate Substrate % inhibition conc. (μm) BoNT/A SNAP > 90 BoNT/B synaptobrevin 20 0 BoNT/D synaptobrevin 20 0 BoNT/E SNAP BoNT/F synaptobrevin 20 0 Thermolysin syn. peptide KLSELDDRADALQAGAS 500 0

44 Inhibition by displacement of catalytic water molecule apo BoNT/A LC BoNT/A LC - p435 DNP-DAB E224 H223 w Zn 2+ E262 E224 Arg sn NH 3 Zn 2+ sc H223 E262 H227 H227

45 Promiscuous binding modes of inhibitors to BoNT/A LC

46 Plasticity of the apo BoNT/A LC protease Three different crystal forms of apo BoNT/A LC Burnett, Stegmann et al. J.Biol.Chem. 2007

47 BoNT/A LC is a very unusual protease Exosites confer substrate specificity between BoNT/A LC protease and SNAP-25 BoNT/A LC captures a partially folded state of SNAP-25 Promiscuous inhibitor binding to active site region Plasticity of BoNT/ LC High affinity p435 inhibitor adopts a very different conformation than SNAP-25 when bound to BoNT/A LC High affinity and specificity of p435 is accomplished by induction of new binding pockets

48 Acknowledgments BoNT/A - SNAP-25 complex Mark Breidenbach BoNT/B - synaptotagmin II complex Rongsheng Jin Thomas Binz, Univ. of Hannover Andreas Rummel, Univ. of Hannover BoNT/A - p435 complex Jorge Zuniga Demet Arac Tim Fenn James Burnett, USARMIID James Schmidt, USARMIID Rick Gussio, USARMIID Robert Stafford, USARMIID Shirin Badie, USARMIID Sina Bavari, USARMIID