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

Sagaya T L Philominathan, Osamu Matsushita, Robert Gensure, Joshua Sakon

Research output: Contribution to journalArticle

15 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)3589-3601
Number of pages13
JournalFEBS Journal
Volume276
Issue number13
DOIs
StatePublished - Jul 2009

Fingerprint

Microbial Collagenase
Collagen
Denaturation
Ions
Calcium
Gas Gangrene
Trifluoroethanol
Peptides
Guanidine
Isomerization
Chelation
Urea
Digestion
Proteins
Hot Temperature
Gases
Association reactions
Tissue
Amino Acids
Enzymes

Keywords

  • Ca
  • Collagen-binding domain
  • Linker
  • Stability
  • Structural transformation

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Ca2+-induced linker transformation leads to a compact and rigid collagen-binding domain of Clostridium histolyticum collagenase. / Philominathan, Sagaya T L; Matsushita, Osamu; Gensure, Robert; Sakon, Joshua.

In: FEBS Journal, Vol. 276, No. 13, 07.2009, p. 3589-3601.

Research output: Contribution to journalArticle

Philominathan, Sagaya T L ; Matsushita, Osamu ; Gensure, Robert ; Sakon, Joshua. / Ca2+-induced linker transformation leads to a compact and rigid collagen-binding domain of Clostridium histolyticum collagenase. In: FEBS Journal. 2009 ; Vol. 276, No. 13. pp. 3589-3601.
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