Ca²⁺-induced linker transformation leads to a compact and rigid collagen-binding domain of Clostridium histolyticum collagenase

Clostridium histolyticum collagenase is responsible for extensive tissue destruction in gas gangrene, and its activity is enhanced by calcium ions. The collagen-binding domain is the minimal segment of the enzyme required for binding to insoluble collagen fibrils and for subsequent collagenolysis. The collagen-binding domain is joined to another binding module by a conserved 14-amino-acid linker. The linker undergoes secondary structural transformation from an α-helix to a β-strand and forms a nonprolyl cis-peptide in the presence of calcium ions. In this study, various biophysical methods were utilized to better understand the structure and functional role of the novel calcium-activated linker. Two Ca²⁺ ions bind cooperatively with macroscopic association constants of K₁ = 5.01 × 10⁵ m^-1 and K₂ = 2.28 × 10⁵ m^-1. The chelation of the second calcium ion is enthalpically unfavorable, which could be a result of isomerization of the nonprolyl cis-peptide. The holo protein is more stable than the apo protein against thermal denaturation (ΔT_m ∼ 20 °C) and chemical denaturation (ΔΔG_H2O ∼ 3 kcal·mol^-1 for urea or guanidine HCl denaturation and Δ20% v/v in 2,2,2-trifluoroethanol). The compact holo collagen-binding domain is more resistant to proteolytic digestion than the apo collagen-binding domain. The orientation of the linker appears to play a crucial role in the stability and dynamics of the collagen-binding domain.