The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium 
Clostridium botulinum are responsible for the disease botulism and include a 
group of immunologically distinct serotypes (A, B, E, and F) that are considered 
to be the most lethal natural proteins known for humans. Two BoNT serotypes, C 
and D, while rarely associated with human infection, are responsible for deadly 
botulism outbreaks afflicting animals. Also associated with animal infections is 
the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a 
hybrid of the BoNT/C (∼two-third) and BoNT/D (∼one-third) serotypes. While the 
amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD 
(BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of 
BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To 
obtain structural insights for the different membrane binding properties, the 
crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å 
resolution and compared to previously reported structures for BoNT/D-HCR. 
Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization 
observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a 
C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with 
BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent 
residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role 
than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt 
bridge with E1247 and may enhance membrane interactions by stabilizing the 
putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the 
surface of the protein. A sulfate ion bound to K1136 may mimic a natural 
interaction with the negatively changed phospholipid membrane surface. 
Liposome-binding experiments demonstrate that BoNT/CD-HCR binds 
phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.