Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic 
disease botulism, is the most poisonous protein known for humans. Produced by 
different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects 
cellular intoxication via a multistep mechanism executed by the three modules of 
the activated protein. Endocytosis, the first step of cellular intoxication, is 
triggered by the ~50 kDa, heavy-chain receptor-binding domain (HCR) that is 
specific for a ganglioside and a protein receptor on neuronal cell surfaces. 
This dual receptor recognition mechanism between BoNT and the host cell's 
membrane is well documented and occurs via specific intermolecular interactions 
with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of 
BoNT-HCR, Hcn, comprises ~50% of BoNT-HCR and adopts a β-sheet jelly roll fold. 
While suspected in assisting cell surface recognition, no unambiguous function 
for the Hcn sub-domain in BoNT has been identified. To obtain insights into the 
potential function of the Hcn sub-domain in BoNT, the first crystal structure of 
a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. 
Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å 
resolution with a tetraethylene glycol (PG4) moiety bound in a hydrophobic cleft 
between β-strands in the β-sheet jelly roll fold of the Hcn sub-domain. The PG4 
moiety is completely engulfed in the cleft, making numerous hydrophilic (Y932, 
S959, W966, and D1042) and hydrophobic (S935, W977, L979, N1013, and I1066) 
contacts with the protein's side chain and backbone that may mimic in vivo 
interactions with the phospholipid membranes on neuronal cell surfaces. A 
sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 
in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar 
protein, BoNT/D-HCR, a sialic acid molecule was observed bound to the equivalent 
residues suggesting that residues T1176, D1177, K1196, and R1243 in BoNT/CD may 
play a role in ganglioside binding.