Crucial Role of the Disulfide Bridge between Botulinum Neurotoxin Light and Heavy Chains in Protease Translocation across Membranes

Abstract

Clostridial botulinum neurotoxins (BoNTs) exert their neuroparalytic action by arresting synaptic exocytosis. Intoxication requires the disulfide-linked, di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol. Here, we investigate the role of the disulfide bridge in the dynamics of protein translocation. We utilize a single channel/single molecule assay to characterize in real time the BoNT channel and chaperone activities in Neuro 2A cells under conditions that emulate those prevalent across endosomes. We show that the disulfide bridge must remain intact throughout LC translocation; premature reduction of the disulfide bridge after channel formation arrests translocation. The disulfide bridge must be on the trans compartment to achieve productive translocation of LC; disulfide disruption on the cis compartment or within the bilayer during translocation aborts it. We demonstrate that a peptide linkage between LC and HC in place of a disulfide bridge is insufficient for productive LC translocation. The disulfide linkage, therefore, dictates the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo. Based on these and previous findings we suggest a sequence of events for BoNT LC translocation to be HC insertion, coupled LC unfolding, and protein conduction through the HC channel in an N to C terminus orientation and ultimate release of the LC from the HC by reduction of the disulfide bridge concomitant with LC refolding in the cytosol. Clostridium botulinum neurotoxins (BoNTs)2 inhibit synaptic exocytosis in peripheral cholinergic synapses, thereby causing flaccid paralysis (1). BoNTs are synthesized as a single polypeptide chain with a molecular mass of ϳ150 kDa. The BoNT polypeptide is then proteolytically cleaved by bacterial or host proteases into the activated di-chain form: an ϳ50-kDa light chain (LC) and an ϳ100-kDa heavy chain (HC). The LC and HC are cross-linked by a disulfide bond between the two chains. Structurally, BoNTs consist of three modules (1-4): The N-terminal LC is the catalytic domain, and the HC comprises the translocation domain (the N-terminal half) and the receptor-binding domain (the C-terminal half). The LCs of six of the seven isoforms of BoNT, designated A-G, have been crystallized and all share structural similarity to the Zn 2ϩ -metalloprotease thermolysin (2, 4 -11). BoNT LCs are sequencespecific endopeptidases that cleave unique components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, the synaptic vesicle fusion complex required for membrane fusion (12-14).BoNTs enter cells by receptor-mediated endocytosis (1, 15). It has been widely recognized that, with the exception of BoNT/D, BoNT entry into neuronal cells requires surface receptors involving a specific ganglioside, namely GT1b (16 -1...

Bimodal modulation of the botulinum neurotoxin protein-conducting channel

“…BoNT/A holotoxin channels were allowed to form in the absence of toosendanin; after onset of low conductance channel activity, toosendanin was added to the trans compartment. Although 0.4 nM toosendanin has no effect on LC translocation, 4 nM toosendanin persistently arrests channel activity at an intermediate step of LC translocation (23,24). Exposure to higher toosendanin concentrations at this early step in translocation progressively inhibits it more effectively and, at 40 M toosendanin, irreversibly blocks translocation (Fig.…”

“…This steady-state ␥ is also a hallmark of isolated HC recorded under identical conditions (22); therefore it represents the conductance of the cargo-free, protein-conducting channel generated after LC translocation is complete (22)(23)(24). We infer that these conductance intermediates, observed as occluded states, correspond to permissible chaperone-cargo conformations populated during protease translocation; concomitantly, the proteinconducting channel progressively conducts more Na ϩ around the polypeptide chain before entering an exclusively ion-conductive state (22)(23)(24). Transformation of an occluded state characterized by low ␥ intermediates into an unoccluded channel with ␥ ϳ 65 pS only occurs after the LC completes translocation.…”

“…A key step in intoxication is the translocation of BoNT LC by the BoNT HC channel (22)(23)(24)(25). We developed an assay to investigate the dynamics of translocation focusing on the interactions between the HC channel/chaperone and its LC cargo for both BoNT/A and BoNT/E serotypes (23,24).…”

“…Using this assay, the translocation process is monitored in real time and at the single-molecule level in excised membrane patches from Neuro 2A cells (23,24). Translocation requires pH 5.3 on the cis compartment, defined as the compartment containing BoNT, and pH 7.0 on the trans compartment, which is supplemented with the membrane nonpermeable reductant TCEP, conditions that emulate those prevalent across endosomes (23,24). Translocation is then observed as a time-dependent increase in Na ϩ conductance (␥) through the HC channel (23,24), as illustrated for BoNT/A by the control experiment shown in Fig.…”

“…We developed an assay to investigate the dynamics of translocation focusing on the interactions between the HC channel/chaperone and its LC cargo for both BoNT/A and BoNT/E serotypes (23,24). Using this assay, the translocation process is monitored in real time and at the single-molecule level in excised membrane patches from Neuro 2A cells (23,24). Translocation requires pH 5.3 on the cis compartment, defined as the compartment containing BoNT, and pH 7.0 on the trans compartment, which is supplemented with the membrane nonpermeable reductant TCEP, conditions that emulate those prevalent across endosomes (23,24).…”