title: Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer.
abstract: We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer.
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
authors.forename: C
authors.initials: C
authors.lastname: Pop
authors.forename: Y R
authors.initials: YR
authors.lastname: Chen
authors.forename: B
authors.initials: B
authors.lastname: Smith
authors.forename: K
authors.initials: K
authors.lastname: Bose
authors.forename: B
authors.initials: B
authors.lastname: Bobay
authors.forename: A
authors.initials: A
authors.lastname: Tripathy
authors.forename: S
authors.initials: S
authors.lastname: Franzen
authors.forename: A C
authors.initials: AC
authors.lastname: Clark
mesh_terms: D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D019281:Dimerization; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity
chemical_list: D061945:Caspase Inhibitors; D004792:Enzyme Precursors; D053148:Caspase 3; D020169:Caspases