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

Abstract

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 (ΔTm ∼ 20 °C) and chemical denaturation (ΔΔGH2O ∼ 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.

Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium histolyticum

“…This may prolong enzyme activity against insoluble collagen in the extracellular matrix. Calcium-bound holo-s3b was previously shown to be more stable than apo-s3b based on DSC and fluorescence spectroscopy-monitored equilibrium denaturation data (11,12). Similar to the observation in s3b, denaturation pathways obtained using either DSC (Fig.…”

“…Meanwhile, the T m value for apo-s3 based on fluorescence data is significantly lower than that determined by DSC (see Table S5 in the supplemental material). The discrepancy in T m was also observed in s3b (12). Analysis of holo-s3 structure using NACCESS (42) demonstrated that Trp925 is solvent inaccessible.…”

“…3). The s3b domain is extremely thermostable (T m ϭ 93°C) despite the torsional strains (12). The s3 domain is only slightly more stable (T m ϭ 94°C).…”

“…When urea was used as the denaturant, the native s3 was exposed to concentrations of denaturant that increased linearly by 0.2 M intervals from 0.0 to 9.8 M. When guanidine hydrochloride (GuHCl) was used as the denaturant, s3 was exposed to concentrations of denaturant that increased linearly by 0.2 M intervals from 0.0 to 5.8 M. During thermal denaturation, s3 was exposed to temperatures that linearly elevated by 2.5°C intervals from 7.5 to 100°C. ⌬G H2O and m values were calculated as described previously (12).…”

“…The linker is coiled as an ␣-helix in the apo form but is unwound to form a parallel ␤-strand in the holo form, which possibly results in domain rearrangement. Two Ca 2ϩ ions bind cooperatively with macroscopic association constants (K 1 ϭ 5.01 ϫ 10 5 M Ϫ1 and K 2 ϭ 2.28 ϫ 10 5 M Ϫ1 ) to trigger the structural transformation (12). Two Ca 2ϩ ions chelate to s3b in close proximity to each other (3.7 Å).…”