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Supplementary online material:

Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F

Rakhi Agarwal, James J. Schmidt, Robert G. Stafford, and Subramanyam Swaminathan

Supplementary figures:

Supplementary figure 1: Superimposition of inh1 on inh2 of BoNT F-inhibitor complex

structures. Inh1, inh2 and BoNT F are shown in ribbon cartoon in red, green and light

gray color.

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1626

Supplementary figure 2: The superposition and comparison of α-helical region 46-54aa

of VAMP (inh1) with the corresponding region in VAMP-2 of SNARE complex (pdb id

1N7S)1. This region folds similar to the hydrophobic residues buried at the interface. The

interacting surfaces complement one another, inh1 with BoNT F, and VAMP-2 with

other members of SNARE complex, SNAP25 and Syntaxin. The interacting residues are

shown in ball and stick model, SNARE-complex SNAP-25 (2 chains) and syntaxin in

orange, VAMP-2 of SNARE complex and inh-1 in cyan and green, respectively.


Supplementary figure 3: Biochemical analysis on various residues of VAMP. The

cleavage of mutant proteins was compared with equimolar quantity of VAMP-native by

2nM BoNT F (1-419). The values are average of three experiments and shown as bars

with standard deviations.


Supplementary figure 4: The cross-eyed stereo view of the superposed structures of

substrate free and substrate bound BoNT F structures. The substrate free (PDB id 2A8A)2

and substrate bound inh1 with BoNT F (PDB id: 3FIE) are in yellow and green color

cartoon, respectively. Only one monomer of inh1 (chain A) is compared. Substrate is not

shown for clarity.


Supplementary figure 5: The conformational difference between substrate-free and

VAMP bound-BoNT F structures. Variations are mostly in side chain conformations.

Substrate-free BoNT F (pdb id 2A8A)2 and BoNT F of complex are shown in yellow and

light gray stick model, respectively. VAMP (inh1) is in green stick model and zinc is in

magenta color.


Supplementary figure 6: VAMP and SNAP-25 binding vs belt in BoNT B and A.

VAMP follows the same direction as SNAP-25 in BoNT A but positions differently in

BoNT F (pdb id 1XTG). The major exosites are significantly different between them and

are presumably the basis for the substrate specificity. SNAP-25 and VAMP bindings

match individually with the belts of BoNT A and B, respectively. BoNT F, Inh1, BoNT

B-belt, BoNT A-belt and SNAP-25 are shown in light gray, blue, green, red and orange

color cartoon.


(a)

(b)


(c)

Supplementary figure 7: The comparison of exosite-1 and exosite-2 of BoNT F with

other VAMP recognizing serotypes, BoNT B, D and G. The figures are in cross-eyed

stereo view. (a) VAMP binding exosites are superposed on BoNT B catalytic domain

(pdb id 1F82). BoNT F, inh1, BoNT B are in cyan, blue and green color cartoon and stick

model. (b) VAMP binding exosites are superposed on BoNT D catalytic domain (pdb id

2FPQ). BoNT F, inh1, BoNT D are in cyan, blue and magenta color cartoon and stick

model. (c)VAMP binding exosites are superposed on BoNT G catalytic domain (pdb id

1ZB7). BoNT F, inh1, BoNT G are in cyan, blue and wheat color cartoon and stick

model.


(a)

(b)

Supplementary figure 8: B value analysis of inhibitor peptides. (a) The inh1 peptide

(bound to molecule A) is represented in sphere model and color coded according to B

values, ranging from 19 Å2 (blue) to 42 Å2 (red) with the average value being 29 Å2

compared to the average B value of 22 Å2 for the enzyme atoms. (b) The inh2 peptide is

represented in sphere model and color coded according to B values, ranging from 39 Å2

(blue) to 51 Å2 (red) with the average value being 47 Å2 compared to the average B value

of 39 Å2 for the enzyme atoms.


Supplementary Discussion

Binding of VAMP to BoNT F Vs belt in BoNT B: In BoNT holotoxins A and B the belt

regions of the translocation domains wrap around and occlude the active site, though it is

more exposed in BoNT B than in BoNT A3,4. Upon reduction of the inter chain disulfide

bond the two domains separate, eliminating contacts with the belt region. The substrate is

presumed to occupy the region vacated by the belt region5. To analyze the relationship

between the belt and the substrate in BoNT F we superimposed four different structures:

BoNT B catalytic domain with its belt region (PDB id 1EPW), BoNT A catalytic domain

with SNAP-25 (pdb id 1XTG), BoNT A-catalytic domain with its belt region (pdb id

3BTA) and BoNT F- inh1 structure. BoNT B structure was chosen for the analysis since

the BoNT F holotoxin structure is not yet available and VAMP is the substrate for BoNT

B also. Interestingly, the overall comparison indicated that SNAP-25 binding is mostly

similar to the belt of BoNT A and VAMP to the belt of BoNT B (Supplementary fig. 6).

However, SNAP-25 and VAMP are positioned differently with respect to each other,

especially at the exosites.

Even though binding of VAMP to BoNT F is relatively similar to the belt of

BoNT B (and presumably of BoNT F), the similarity is pronounced for N25-Q38, the

region almost before the V1-SNARE motif binding (Supplementary Fig. 6). Importantly,

the main chain β-sheet interactions as seen for exosite-3 are similar to belt and BoNT B,

and VAMP and BoNT F. However, exosite-1, exosite-2 and the active site interactions

are exclusive features that lead to a difference in binding of the two. This suggests that

even though VAMP might bind and replace the belt region of the translocation domain in

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various serotypes (B,D,F,G and tetanus), the major substrate recognition and selection is

through exosites unique for each serotype and also through S2 and S1’ pockets.

Implications for VAMP recognition and binding by other serotypes: BoNTs B, D, F,

G and tetanus neurotoxin cleave VAMP6. BoNT B and tetanus cleave the peptide bond

Q76-F77 and BoNT G cleaves A81-A82. Interestingly, BoNT F and D cleave adjacent

peptide bonds, Q58-K59 and K59-L60, respectively. We have shown in our earlier

structural studies proper docking of the substrate P1’ residue in the S1’ pocket forms the

basis for specificity with regard to the scissile bond to be cleaved7, accordingly the S1’

pocket should be different to accommodate L60 (P1’) for BoNT D. Exosites, S2 and S1’

pockets dictate the specificity of scissile bond by a serotype.

Interesting differences were observed when we superimposed the catalytic

domains of BoNT B (pdb id 1F82), D (pdb id 2FPQ), G (pdb id 1ZB7) 7-10 and BoNT F-

inh1. The loop residues 171-180 of BoNT F mainly contribute to the hydrophobic

pockets for the exosite-1 and -2, in BoNT F-inh1 and -inh2 structures (supplementary

figure 7a, b and c). This loop is relatively small in serotypes B, D and G and most likely

will not allow similar binding of VAMP at this position. The V1-SNARE docking

regions as in BoNT F are not conserved in all three. Also, the salt bridge and ionic

interactions of non-conserved Arg133 and Arg171 are missing in all other serotypes. This

suggests a difference in VAMP recognition among these serotypes.

BoNT D cleaves the K59-L60 peptide bond adjacent to BoNT F scissile bond.

However, when BoNT F-inh1 structure was superimposed on BoNT D, P1-P4 of the

substrate peptide takes the similar position as BoNT D residues 169-172. This may be

because loop 167-174 is disordered in BoNT D with some residues not being modeled. In

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view of this, comparison near this region may not be reliable and we restrict our

comparison to the rest of the substrate peptide. The differences in hydrophobic patch for

exosite-1 and variation of residues in exosite-2 between BoNT F and D suggest a

difference in binding and thus shifting of the scissile bond. The structural comparison

suggests that in BoNT D the VAMP residues forming the helical region (47-54) might

shift towards the exosite-2 (of BoNT F) and the V1-SNARE motif might shift towards

exosite-3 (of BoNT F) and possibly be stabilized by the interactions with the 150 loop

through hydrophobic interactions. VAMP in BoNT D overall might shift more than 3-4

residues from exosite-1 towards exosite-2 than by one residue shift. The differences

basically are due to unconserved exosite-1 and -2 residues (of BoNT F) between them

and as mentioned earlier, BoNT D has a smaller loop than 171-180 of BoNT F. Also,

since we did not observe any major change in main chain conformation at exosite-1 and -

2 in BoNT F upon VAMP binding, presumably similar restrictions in BoNT D will lead

to a different mode of VAMP binding than BoNT F. However a structural study on BoNT

D-VAMP complex will unravel the details. We propose that the binding of the residues

P1-P4 in BoNT D will be similar to BoNT F.

References:

1. Ernst, J.A. & Brunger, A.T. High resolution structure, stability, and

synaptotagmin binding of a truncated neuronal SNARE complex. J. Biol. Chem.

278, 8630-6 (2003).

2. Agarwal, R., Binz, T. & Swaminathan, S. Structural analysis of botulinum

neurotoxin serotype f light chain: implications on substrate binding and inhibitor

design. Biochemistry 44, 11758-65 (2005).

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3. Lacy, D.B., Tepp, W., Cohen, A.C., DasGupta, B.R. & Stevens, R.C. Crystal

structure of botulinum neurotoxin type A and implications for toxicity. Nat.

Struct. Biol. 5, 898-902 (1998).

4. Swaminathan, S. & Eswaramoorthy, S. Structural analysis of the catalytic and

binding sites of Clostridium botulinum neurotoxin B. Nat. Struct. Biol. 7, 693-699

(2000).

5. Brunger, A.T. et al. Botulinum neurotoxin heavy chain belt as an intramolecular

chaperone for the light chain. PLoS Pathog. 3, 1191-4 (2007).

6. Montecucco, C. & Schiavo, G. Mechanism of action of tetanus and botulinum

neurotoxins. Mol. Microbiol. 13, 1-8 (1994).

7. Agarwal, R. & Swaminathan, S. SNAP-25 substrate peptide (residues 180-183)

binds to but bypasses cleavage by catalytically active Clostridium botulinum

neurotoxin E. J. Biol. Chem. 283, 25944-51 (2008).

8. Arndt, J.W., Chai, Q., Christian, T. & Stevens, R.C. Structure of botulinum

neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2

substrate specificity. Biochemistry 45, 3255-62 (2006).

9. Arndt, J.W., Yu, W., Bi, F. & Stevens, R.C. Crystal structure of botulinum

neurotoxin type G light chain: serotype divergence in substrate recognition.

Biochemistry 44, 9574-80 (2005).

10. Hanson, M.A. & Stevens, R.C. Cocrystal structure of synaptobrevin-ll bound to

botulinum neurotoxin type B at 2.0 A resolution. Nat. Struct. Biol. 7, 687-692

(2000).

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