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. 2014 Nov 11;426(22):3773-3782.
doi: 10.1016/j.jmb.2014.09.009. Epub 2014 Sep 18.

Structural basis of the pH-dependent assembly of a botulinum neurotoxin complex

Tsutomu Matsui  1 , Shenyan Gu  2 , Kwok-Ho Lam  2 , Lester G Carter  3 , Andreas Rummel  4 , Irimpan I Mathews  3 , Rongsheng Jin  5
Affiliations

Structural basis of the pH-dependent assembly of a botulinum neurotoxin complex

Tsutomu Matsui et al. J Mol Biol. .

Abstract

Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH.

Keywords: X-ray scattering; botulinum neurotoxin; neurotoxin-associated proteins; non-toxic non-hemagglutinin; progenitor toxin complex.

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Figures

Fig. 1
Fig. 1. Structures of the M-PTC, BoNT/Ai, and NTNHA-A
(A) Surface representation of the M-PTC with each domain labeled in a distinct color (PDB ID: 3V0A) [8]. The same color code and view angles are used in all the following figures. (B–C) The structures of BoNT/Ai and NTNHA-A in the context of the M-PTC. (D) BoNT/A in the free form (PDB ID: 3BTA) [18]. Note that the HC domain is labeled in light green or purple in the complex form or the free form, respectively.
Fig. 2
Fig. 2. SAXS studies of BoNT/Ai
(A) Scattering profiles of BoNT/Ai at pH 8.0 (purple) and 6.0 (green). (i) Curve fitting of data at pH 8.0 with the crystal structure of free form BoNT/A. (ii) Curve fitting of data at pH 8.0 with the SAXS model determined by rigid body refinement. (iii) Superimposition of SAXS profiles at pH 8.0 and 6.0. Constant subtractions used in the rigid body refinements were taken into account. (iv) Curve fitting of data at pH 6.0 with the crystal structures of BoNT/A in the free (solid line) and the complex (dash line) forms. (v) Curve fitting of data at pH 6.0 with the SAXS model determined by rigid body refinement. All curve fittings were performed using the program CORAL [20]. (B) Kratky plots of the experimental profiles and theoretical curve of BoNT/Ai. (C) Pair distance distributions P(r) of BoNT/Ai at pH 8.0 and 6.0. Theoretical curves are shown as well. (D) The SAXS model of BoNT/Ai at pH 8.0 determined by rigid body refinement. The HC domain was refined as a rigid body (shown as brown tubes). Crystal structure of the free form BoNT/A is superimposed onto the LC–HN domain, with the HC domain labeled in purple tubes. The position of reconstructed loop is indicated by an arrow. (E) The SAXS model of BoNT/Ai at pH 6.0 with the HC domain labeled in cyan. (F) Comparison of the SAXS models between pH 8.0 and 6.0.
Fig. 3
Fig. 3. SAXS studies of NTNHA-A
(A) Scattering profiles of NTNHA-A at pH 6.0 (red) and 8.0 (blue). (i) Curve fitting with the crystal structure in the M-PTC (PDB ID: 3V0A, chain B). (ii) Curve fitting with the SAXS model determined by rigid body refinement. (iii) Superimposition of SAXS profiles between pH 8.0 and 6.0. Constant subtractions used at the rigid body refinements were taken into account. A close up view at q = 0.05–0.125 Å-1 is shown in the inset. (iv) Curve fitting with the crystal structure (PDB ID: 3V0A, chain B). (v) Curve fitting with the SAXS model determined by rigid body refinement. (B) Kratky plots of NTNHA-A at pH 8.0 and 6.0. Theoretical curve of the crystal structure is also shown. (C) Pair distance distributions P(r) of NTNHA-A at pH 8.0 and 6.0. Theoretical curve is shown as well. Peak positions are significantly different. (D) The SAXS model of NTNHA-A at pH 6.0 determined by rigid body refinement (shown as gray tubes). The nHC domain was refined as rigid body while two disordered loops in the crystal structure were reconstructed. The crystal structure is superimposed onto the nLC–nHN domain and the nHC domain is shown in red tube. The positions of reconstructed loops are indicated by arrows. (E) The SAXS model of NTNHA-A at pH 8.0 (gold tube). (F) Comparison of SAXS models between pH 6.0 and 8.0.
Fig. 4
Fig. 4. FPLC-SAXS studies of the M-PTC
(A) Gel-filtration profile and Rg values in the online FPLC-SAXS experiment. The profile was made by plotting X-ray intensity of each image. The real and reciprocal space Rgs were estimated by the program AUTOGNOM [36]. (B) SAXS profile of the M-PTC (yellow) and curve fitting with the crystal structure. Kratky plot is shown in inset. (C) An ab-initio model derived from the SAXS profile. The most populated volume in solution, which was filtered using the program DAMFILT in DAMAVER suite [38] is displayed in gray.
Fig. 5
Fig. 5. A schematic representation of the assembly of the M-PTC
BoNT/A and NTNHA-A are predominantly monomeric and each adopts a relatively rigid conformation at pH 8.0, which is unable to form a complex. Decreasing the environmental pH to 6.0 does not significantly change the conformation of BoNT/A. However, it induces a conformational change of the nHC domain of NTNHA-A, which is then slightly splayed out to initiate interactions with BoNT/A. At the meantime, the acidic pH protonates pH-sensing residues on BoNT/A and NTNHA-A, which strengthen the inter-molecule interactions. Subsequently, a mutual induced fit triggers a large-reorientation of the HC domain of BoNT/A while the nHC domain of NTNHA-A undergoes a further conformational change to tightly lock with BoNT/A.
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