title: 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 alpha-helix to a beta-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 Ca(2+) ions bind cooperatively with macroscopic association constants of K(1) = 5.01 x 10(5) m(-1) and K(2) = 2.28 x 10(5) 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 (DeltaT(m) approximately 20 degrees C) and chemical denaturation (DeltaDeltaG(H2O) approximately 3 kcal x mol(-1) for urea or guanidine HCl denaturation and Delta20% 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. pstleena@uark.edu
authors.forename: Sagaya T L
authors.initials: ST
authors.lastname: Philominathan
authors.forename: Osamu
authors.initials: O
authors.lastname: Matsushita
authors.forename: Robert
authors.initials: R
authors.lastname: Gensure
authors.forename: Joshua
authors.initials: J
authors.lastname: Sakon
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D003012:Microbial Collagenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D014508:Urea / Q000737:chemistry
chemical_list: D014508:Urea; D003094:Collagen; D003012:Microbial Collagenase; D002118:Calcium