Effects of Selective Substitution of Cysteine Residues on the Conformational Properties of Chlorotoxin Explored by Molecular Dynamics Simulations

Chlorotoxin (CTX) is a 36–amino acid peptide with eight Cys residues that forms four disulfide bonds. It has high affinity for the glioma-specific chloride channel and matrix metalloprotease-2. Structural and binding properties of CTX analogs with various Cys residue substitutions with l-α-aminobutyric acid (Abu) have been previously reported. Using 4.2 µs molecular dynamics, we compared the conformational and essential space sampling of CTX and analogs with selective substitution of the Cys residues and associated disulfide bonds with either Abu or Ser. The native and substituted peptides maintained a high degree of α-helix propensity from residues 8 through 21, with the exception of substitution of the Cys(5)–Cys(28) residues with Ser and the Cys(16)–Cys(33) residues with Abu. In agreement with previous circular dichroism spectropolarimetry results, the C-terminal β-sheet content varied less from residues 25 through 29 and 32 through 36 and was well conserved in most analogs. The Cys(16)–Cys(33) and Cys(20)–Cys(35) disulfide-bonded residues appear to be required to maintain the αβ motif of CTX. Selective substitution with the hydrophilic Ser, may mitigate the destabilizing effect of Cys(16)–Cys(33) substitution through the formation of an inter residue H-bond from Ser(16):OγH to Ser(33):OγH bridged by a water molecule. All peptides shared considerable sampled conformational space, which explains the retained receptor binding of the non-native analogs.