title: Structural and functional analysis of botulinum neurotoxin subunits for pH-dependent membrane channel formation and translocation.
abstract: The structure-function relationship of Botulinum Neurotoxin (BoNT) proteins is greatly influenced by pH. While the low pH of endosome favors membrane interaction of the heavy chain (HC) for the formation of a membrane channel and translocation of the light chain (LC), the catalytic activity of the LC requires a neutral pH for cleavage of the soluble NSF attachment protein receptor (SNARE) complex in the cytosol. In this study, we monitored secondary structural characteristics of LC, HC and holotoxin at individual pHs 4.5 and 7.2 and at the transition pH4.5 to 7.2 to identify the structural signatures underlying their function. The HC showed higher thermal stability at pH4.5 with a melting temperature (Tm) of 60.4°C. The structural analysis of HC in the presence of liposomes showed no difference in ellipticity with that of HC at pH7.2 at 208 and 222 nm but a 25.2% decrease in ellipticity at 208 nm at acidic pH, indicating low pH-induced structural changes that might facilitate interaction with the membrane. Further, HC showed 18% release of K+ ions from liposomes at pH4.5 as against 6% at neutral pH, reinforcing its role in membrane channel formation. LC on the other hand, showed maximum ellipticity at pH7.2, a condition that is relevant to its endopeptidase activity in the cytosol of the neurons. Also, the similarity in the structures at pH7.2 and transition pH4.5 to 7.2 suggested that the flexibility acquired by the protein at low pH was reversible upon exposure to neutral pH for cleavage of SNARE proteins.
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA.
authors.forename: Gowri
authors.initials: G
authors.lastname: Chellappan
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA; Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA 02747, USA.
authors.forename: Raj
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Santos
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Lowell, Lowell, MA 01854, USA.
authors.forename: Dipak
authors.initials: D
authors.lastname: Goyal
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA.
authors.forename: Shuowei
authors.initials: S
authors.lastname: Cai
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA; Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA 02747, USA; Prime Bio, Inc., Dartmouth, MA 02747, USA. Electronic address: bsingh@inads.org.
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003014:Clostridium botulinum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D017136:Ion Transport; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D050600:SNARE Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D007473:Ion Channels; D008081:Liposomes; D021122:Protein Subunits; D050600:SNARE Proteins; D001905:Botulinum Toxins; D011188:Potassium
keywords: BoNT; Circular dichroism; Clostridium botulinum; Heavy chain; Light chain; Molar ellipticity; Thermal denaturation; pH