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. Author manuscript; available in PMC: 2014 Dec 1.
Published in final edited form as: Toxicon. 2013 Feb 5;0:101–107. doi: 10.1016/j.toxicon.2013.01.007

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function

Audrey Fischer 1,1, Mauricio Montal 1,*
PMCID: PMC3797153  NIHMSID: NIHMS443036  PMID: 23396042

Abstract

Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication.

Keywords: Protein translocase, Biological weapons, Botulism, Antibotulinal agents, Modular nanomachine

1. Introduction

Botulinum neurotoxin (BoNT) inhibits synaptic exocytosis in peripheral cholinergic synapses causing botulism, a disease characterized by descending flaccid paralysis. Clostridium botulinum cells secrete seven BoNTs isoforms designated as BoNT/A to G (Schiavo et al., 2000). All BoNT isoforms are synthesized as a single polypeptide chain and then cleaved to form a disulfide linked di-chain molecule by either clostridial or host cell proteases (Sathyamoorthy and DasGupta, 1985). The mature holotoxin consists of a 50 kDa light chain (LC) protease and a 100 kDa heavy chain (HC). The HC is comprised of the translocation domain (TD) (the N-terminal half), a long four-helix bundle, and the receptor-binding domain (RBD) (the C-terminal half), consisting of a single β-barrel and a single β-trefoil motif (Lacy and Stevens, 1999; Lacy et al., 1998; Schiavo et al., 2000; Swaminathan and Eswaramoorthy, 2000).

BoNT enter neurons by receptor-mediated endocytosis, initiated by the interactions between the BoNT RBD and a specific ganglioside, GT1B (Ginalski et al., 2000; Nishiki et al., 1996; Rummel et al., 2009; Tsukamoto et al., 2005; Yowler et al., 2002) and protein co-receptor, SV2 for BoNT/A (Dong et al., 2006; Mahrhold et al., 2006), /D (Peng et al., 2011), /E (Dong et al., 2008) and /F (Fu et al., 2009) and synaptotagmins I and II for BoNT/B and BoNT/G (Rummel et al., 2004). Exposure of the BoNT-receptor complex to the acidic environment of endosomes induces a conformational change whereby the HC inserts into the endosomal bilayer membrane (Finkelstein, 1990; Gambale and Montal, 1988; Schiavo et al., 2000). Previously, we demonstrated that the HC forms a protein-conducting channel under endosomal conditions (Fischer and Montal, 2006), and translocates the LC protease into the cytosol (Fischer and Montal, 2007b; Koriazova and Montal, 2003), colocalizing it with its substrate SNARE (soluble NSF attachment protein receptor) protein (Blasi et al., 1993; Schiavo et al., 1992, 2000). Effectively, the HC functions as a protein translocase.

The LC and the C-terminal half of the RBD crystals have been solved for multiple serotypes and their functions clearly defined (Agarwal et al., 2005, 2004; Arndt et al., 2006, 2005; Breidenbach and Brunger, 2004; Chai et al., 2006; Hanson and Stevens, 2000; Jin et al., 2006; Segelke et al., 2004); however the central motifs of the protein are less well understood. The recent crystal structures of the BoNT/B RBD C-terminal half interacting with its ganglioside and protein co receptors raises queries regarding the role of the N-terminal half of the RBD (Chai et al., 2006; Jin et al., 2006). Potentially, the N-terminal motif of the RBD could serve a function in priming the TD to an insertion competent orientation with respect to the membrane (Muraro et al., 2009), or function as part of the protein-conducting channel itself. The elongated, unstructured belt region of the TD is another domain with an elusive function (Brunger et al., 2007; Galloux et al., 2008); potentially acting as a pseudo-substrate/chaperone for the LC during the majority of the intoxication process (Brunger et al., 2007).

Molecular dissection allowed us to investigate the domain requirements for BoNTchannel activity and protein translocation. By utilizing the single molecule LC translocation assay previously developed in our lab, we were able to determine the minimal module required to: 1. elicit BoNT ion channel activity, and 2. productively translocate LC. The assay allowed us to examine the pH threshold necessary for each of these functions as well as the pivotal role of the inter-chain disulfide bridge in the translocation process.

2. Minimal channel forming unit

We examined the channel activity of several sub-domains of BoNT/A holotoxin: HC, N-terminal half of the HC called the translocation domain (TD), and the TD without the N-terminal 100 amino acids known as the belt region (BTD). Protein insertion and channel formation were monitored on excised membrane patches from Neuro 2A cells under conditions which recapitulate those across endosomal membranes: the cis compartment, containing BoNT/A protein, was held at pH 5.3 and the trans compartment was maintained at pH 7.0 and supplemented with the membrane nonpermeable reductant TCEP. Channel activity of the TD and BTD modules exhibited similar characteristics to HC, as illustrated in Fig. 1. Single channel conductance (γ) was determined to be approximately 65 pS as measured from the transition from the closed (C) to open (O) states; channel activity occurred in bursts between quiescent periods. The channel transitions through a 10 pS subconductance state at the onset and exit from a burst; within a burst the channel undergoes quick transitions between the intermediate state and the open state. The probability to reside in the open state (Po), measured from the ms timescale transitions between the open and closed states, of each protein were all indistinguishable from that of BoNT/A HC as illustrated by the time expansion of current records shown in Fig. 1. The TD and BTD have similar voltage dependence to the unoccluded state exhibited by the BoNT/A HC channel after completion of translocation (Fischer and Montal, 2006, 2007a, 2007b; Fischer et al., 2008, 2012); V1/2, the voltage at which Po = 0.5, is approximately −67 mV for holotoxin, approximately −64 mV for TD, and approximately −65 mV for BTD. Therefore, the BTD embodies a minimum channel forming entity (Fischer et al., 2012).

Fig. 1.

Fig. 1

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A – BoNT/A HC (Fischer and Montal, 2006), B – TD (Fischer et al., 2008), and C – BTD (Fischer et al., 2012) channel activity measured on excised patches of Neuro 2A cells. Representative single-channel currents at the indicated voltages; consecutive voltage pulses applied to the same patch for each experimental condition. Channel opening is indicated by a downward deflection; C and O denote the closed and open states. γ values for HC = 65.3 ± 0.4 pS, TD = 64.4 ± 0.4 pS, and BTD = 69.2 ± 0.9 pS. The sections of the recordings obtained at −110 mV delimited by the red bars are shown in the bottom traces at a 10-fold higher time resolution; the prototypical square events which are characteristic of unitary channel currents are clearly discerned.

3. Channel insertion is pH independent in the absence of the RBD

Previous studies have shown that BoNT forms pH dependent and voltage dependent channels in lipid bilayers (Blaustein et al., 1987; Donovan and Middlebrook, 1986; Hoch et al., 1985) and neuronal cells (Fischer and Montal, 2007b; Sheridan, 1998); however, the pH threshold for channel formation of the TD and BTD was undetermined. In contrast to HC, channel activity of similar characteristics was monitored for the TD and BTD when the cis compartment solution was adjusted to more neutral pH values; representative records are shown in Fig. 2. Bursting patterns and γ was determined to be pH gradient independent (Fig. 2). The voltage dependence of the Po was modulated by pH 6 but not pH 7 in the cis compartment as shown in the lower panel graphs of Fig. 2. The TD interaction with the RBD therefore alters the pH threshold for membrane insertion; in the absence of the RBD the TD and BTD readily form channels at neutral pH.

Fig. 2.

Fig. 2

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A – BoNT/A TD and B – BTD channel activity is independent of a pH gradient across the membrane. Top panels illustrate representative channel activity monitored with ΔpH 5 cis/7 trans, middle panel shows ΔpH 6/7 and bottom panel shows no ΔpH, symmetric pH 7, all measured at an applied voltage of −100 mV. After GΩ seal formation, TD channel activity begins at 30 min (top), 14 min (middle), and 30 min (bottom) and BTD channel activity begins at 14 min (top), 18 min (middle) and 29 min (bottom). Low conductance intermediates were not observed. Representative 200 ms segments of long recordings are illustrated; the single channel conductance for each condition is indicated. Corresponding lower panels display analysis of the voltage-dependence of unoccluded channel activity for BoNT/A TD for ΔpH 5/7 ( Inline graphic) (V1/2 = −64.0 ± 4.2 mV), ΔpH 6/7 ( Inline graphic) (V1/2 = −46.8 ± 3.1 mV), ΔpH 7/7 ( Inline graphic) (V1/2 = −55.3 ± 3.8 mV), and for BTD for ΔpH 5/7 ( Inline graphic) (V1/2 = −64.9 ± 2.2 mV), ΔpH 6/7 ( Inline graphic) (V1/2 = −29.4 ± 4.2 mV), and ΔpH 7/7 ( Inline graphic) (V1/2 = −58.9 ± 9.0 mV) (Fischer et al, 2008, 2012).

4. Minimal protein translocation unit

Translocation of BoNT/A LC and BoNT/E LC through the HC channel has been consistently monitored in real time and at the single molecule level in excised membrane patches from Neuro 2A cells (Fischer and Montal, 2007b). Similar results were recently reported for BoNT/B (Sun et al., 2011). For BoNT/A, LC translocation is observed as a time-dependent increase in Na+ conductance through the HC channel; the initial 14 pS channel activity rapidly transitions through several intermediate γ before reaching a stable γ approximately 68 pS (Fischer and Montal, 2007a, 2007b). The steady-state γ is also the characteristic conductance of isolated HC recorded under identical conditions; therefore it represents the conductance of the unoccluded HC in holotoxin experiments after translocation is complete. We interpret the intermediate conductance states as reporters of discrete transient steps during the translocation of the LC across the membrane. During protease translocation, the protein-conducting channel progressively conducts more Na+ around the polypeptide chain before entering an exclusively ion-conductive state. This typical pattern of channel activity for holotoxin proceeds under conditions which mimic those across endosomes and lead to LC translocation and retrieval of protease activity after completion of translocation.

We examined the role of the disulfide bridge during translocation by modulating the redox gradient across the membrane during LC translocation. In the absence of a reducing environment in the trans compartment, BoNT/A holotoxin channels were arrested in an intermediate conductance state, never achieving the unoccluded state (Fischer and Montal, 2007a, 2007b). We concluded that without the disulfide bridge reduction, the LC remains locked within the HC channel, unable to escape and refold within the cytosol. In contrast, pre-reduction of holotoxin before assay resulted in HC like channels without intermediates associated with LC translocation: The LC loses association and does not undergo translocation across the membrane (Fischer and Montal, 2007a). If the disulfide bridge is required for initiation of productive translocation, at what stage during translocation must reduction occur in order to productively release the LC into the cytosol? Addition of the membrane permeable beta-mercaptoethanol (βME) to the trans compartment allowed us to rapidly reduce both the cis and trans compartments during each step of LC translocation. Addition of βME at any of the intermediate conductance states resulted in a persistent intermediate state, never achieving the unoccluded HC like state (Fischer and Montal, 2007a). The disulfide bridge therefore must remain intact during LC translocation; entry of the disulfide bridge into the reducing environment of the cytosol serves as the final step required for productive release. Recently, Montecucco et al. have confirmed the crucial role of the disulfide link (Simpson et al., 2004) using an elegant assay in cerebellar granular neurons (Pirazzini et al., 2011).

To investigate the potential role of the receptor binding domain in LC translocation we analyzed the LC-TD channel at early time points of channel activity (Fischer et al., 2008). Under endosomal conditions, LC-TD channel activity was similar to holotoxin; low conductance intermediate states were visualized prior to the unoccluded state (Fig. 3). At the onset of LC-TD channel activity, small, discrete events with a γ approximately 17 pS are clearly discerned. Progressively, γ undergoes a continuous increase until reaching a stable value of 69 pS (Fig. 3A and C red circle). The average time course of Δγ occurs with a t1/2 approximately 130 s and displays multiple discrete transient intermediate conductances before achieving the steady state 69 pS. Productive translocation by LC-TD across cellular membranes was confirmed using a cellular intoxication assay (Eubanks et al., 2007). LC-TD exhibited similar growing γ patterns at pH 6 cis/pH 7 trans (ΔpH 6/7) (Fig. 3A, middle panel). The t1/2, measured from the average time course of change of γ after insertion, is increased from that of pH 5 cis conditions to 190 ± 10 s (Fig. 3A, middle panel, and 3C purple circle). The longer time required to complete LC translocation may correlate with the extent of fold the LC maintains at pH 6. Previous work has demonstrated that bafilomycin, a proton pump inhibitor which neutralizes endosomal compartments, effectively aborts BoNT intoxication (Keller et al., 2004; Simpson et al., 1994; Wang et al., 2008). A more complex set of requirements may underlie the adoption of a conformation of the LC compatible with productive entry and translocation through the channel. Consistent with this model are the results for LC-TD channel activity elicited at symmetric neutral pH; channel activity initiated and maintained at the unoccluded state of 64 pS (Fig. 3A, lower panel, and 3C blue circle). Circular dichroism analysis of the LC at pH 7 indicates a high α-helical content implying that the protein would remain folded rather than adopt a translocation competent conformation (Koriazova and Montal, 2003). We interpret that LC does not unfold at pH 7 and therefore cannot be translocated through the approximately 15 Å pore of the TD channel (Hoch et al., 1985; Smart et al., 1997). Together, these findings substantiate the notion that the LC-TD is a minimum protein translocation unit (Fischer et al., 2008).

Fig. 3.

Fig. 3

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BoNT/A channel activity in excised patches of Neuro 2A cells over range of pH values in the cis compartment. A – BoNT/A LC-TD channel activity begins 10 min after GΩ seal formation, t = 0 s, and transitions from low conductance intermediate state to the unoccluded state after completion of LC translocation. LC-TD channel activity begins 12 min after GΩ seal formation for ΔpH 6/7 and 45 min after seal formation for ΔpH 7/7. B – BoNT/A holotoxin channel activity begins 10 min after GΩ seal formation. Schematic representation of the time course of translocation are depicted under the experimental current recordings; the vertical bars denote interruption of the record to highlight the pattern of activity at the onset of translocation (occluded state) and completion of translocation (unoccluded state). C – Average time course of conductance change for BoNT/A holotoxin DpH 5/7 (●), HC ΔpH 5/7 ( Inline graphic), LC-TD ΔpH 5/7 ( Inline graphic), LC-TD ΔpH 6/7 ( Inline graphic), and LC-TD ΔpH 7/7 ( Inline graphic). No channel activity was monitored for BoNT/A holotoxin under the following experimental conditions: ΔpH 6/7 (□) and ΔpH 7/7 (Δ). D –Analysis of unoccluded channel activity for BoNT/A holotoxin DpH 5/7 (●)(V½ = −67.2 ± 2.9 mV), BoNT/A LC-TD for ΔpH 5/7 ( Inline graphic) (V½ = −59.0 ± 9.1 mV), BoNT/A LC-TD for ΔpH 6/7 ( Inline graphic) (V½ = −28.1 ± 4.5 mV), BoNT/A LC-TD for DpH 7/7 ( Inline graphic) (V½ = −64.1 ± 2.9 mV).

5. Productive intoxication requires several steps of interdomain chaperone activity

The findings summarized here illustrate the modular nature of BoNT, a protein in which each component functions individually yet the tight interplay governs that each domain serves as a chaperone for the others. The RBD insures LC unfolding to a translocation competent conformation occurs in synchrony with TD channel formation (Fig. 3) (Fischer et al., 2008). A similar requirement for BoNT/B has recently been presented by Sun et al. (2011). Sun et al. (2011) further suggested that, for BoNT/B, RBD association with its ganglioside receptor promotes channel formation through oligomerization. The LC chaperones the HC by maintaining it in a soluble conformation at neutral pH until residence within the acidic endosome where the LC serves to gate the TD channel in order to initiate LC translocation (Fischer and Montal, 2007b). The TD belt protects the LC from premature cleavage of non-specific substrates until localization within the cytosol (Brunger et al., 2007). Although the belt is not required for reconstitution of ion channel activity it may facilitate productive LC unfolding to a translocation competent conformation within the acidic compartment of the endosome, a hypothesis supported by the requirement for an intact disulfide bridge between the belt and the LC during LC translocation (Fischer and Montal, 2007a). Finally, the TD protects the LC within the acidic environment of the endosome, chaperones the LC to the cytosol, and releases the LC in an enzymatically active conformation to act on the substrate SNARE proteins (Koriazova and Montal, 2003).

This modular function makes BoNT and similar modular toxins a tool for biomolecule delivery to predetermined target tissues (Bade et al., 2004; Dobrenis et al., 1992; Duggan et al., 2002; Francis et al., 1995; Ichinose et al., 2002; Liu et al., 1999). Proteins of choices have been tethered to enzymatically inactive BoNT and demonstrated to translocate and function within the neuronal cell (Bade et al., 2004). While these studies have limited themselves to modulation of the enzymatic domain, replacement of the neuronal targeting receptor binding domain with one that recognizes a unique cellular surface protein could make BoNT an attractive delivery system for fold permissive cargo proteins to the scientist's target tissue of choice (Edupuganti et al., 2012; Stancombe et al., 2012).

Acknowledgments

The research summarized here was conducted in collaboration with Darren Mushrush, Shilpa Sambashivan, Borden Lacy and Axel Brunger. The original publications have been accordingly cited. This work was supported by the Pacific Southwest Regional Center of Excellence NIH AI065359 and, in part, by NIH-GM49711.

Abbreviations

βME

beta-mercaptoethanol

ΔpH

pH gradient across the cis and trans compartments

γ

single channel conductance

BoNT

botulinum neurotoxin

BTD

beltless translocation domain

HC

heavy chain

LC

light chain

LC-TD

light chain-translocation domain

Po

channel open probability

RBD

receptor binding domain

t1/2

the half time for completion of translocation

TCEP

tris-(2-carboxyethyl) phosphine

TD

translocation domain

V½

the voltage at which Po = 0.5

Footnotes

Ethical statement: The authors declare that the research reported in the above mentioned article was conducted according to the ethical guidelines pertaining to our profession and underscored by the institution.

Conflict of interest: The authors declare that there are no conflicts of interest.

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