Figure 1.
The β6 Strand Is the Major Determinant for the Dimerization of Caspases
(A) Comparison of the dimerization interfaces of caspase-3 and caspase-9. Caspases-3 and -9 exist primarily as a dimer and a monomer, respectively, in solution. However, inhibitor-bound caspase-9 was crystallized in its dimeric form [19]. The overall structures of caspases-3 and -9 are similar. A close-up view of the dimerization interfaces reveals sharp variation of residues on the β6 strand between caspase-3 and -9, which likely contributes to their different propensity for dimer formation. For caspase-9, Phe404 on strand β6 appears to impede dimerization as it sterically clashes with Phe404′ of the adjacent monomer [19].
(B) A sequence alignment of the residues on and surrounding the strand β6 in four representative caspases. Similar to caspase-3, caspases-6 and -7 also exist as homodimers in solution. Residues on β6 are highlighted in blue and orange for the monomeric and dimeric caspases, respectively. Note that the variation of residues on β6 strand appears to correlate with the propensity for dimerization.
Figure 2.
The Dimeric Caspase-9 Exists as a Monomer in Solution
The apparent molecular masses for the WT and dimeric caspase-9 (full-length, residues 1–416) were analyzed by gel filtration. Relevant peak fractions were visualized by SDS-PAGE followed by Coomassie staining. The dimeric caspase-9 was eluted from the column with a molecular mass about twice that of the WT protein.
Table 1.
Analytical Ultracentrifugation Analysis of Caspase-9 Proteins
Figure 3.
The Dimeric Caspase-9 Closely Resembles the WT Caspase-9
(A) Overall structure of the dimeric caspase-9 (C287S). The structural core is shown in blue; the solvent-exposed active site loops are shown in magenta. The β6 and β6′ strands are highlighted in red. Note the asymmetry of the dimer.
(B) Structural superposition of the dimeric caspase-9 (blue and magenta) and the WT caspase-9 (grey and green). The only significant structural difference is in the solvent-exposed active site loops, which is due to the inactive nature of the dimeric caspase-9 (C287S).
(C) A stereo comparison of the region surrounding strands β6 and β6′ in WT and dimeric (engineered) caspase-9. The side chains of the WT and dimeric caspase-9 are shown in orange and yellow, respectively. To avoid congestion in the graphic, residues from only one caspase-9 monomer are labeled. The Cys287 in the upper left corner is the catalytic residue in the active site of the asymmetric caspase-9.
(D) A stereo comparison of the region surrounding strands β6 and β6′ in caspase-3 and dimeric (engineered) caspase-9. The side chains of the caspase-3 and dimeric caspase-9 are shown in green and yellow, respectively. The labels refer to amino acids in caspase-3.
Table 2.
Data Collection and Statistics from Crystallographic Analysis
Figure 4.
The Dimeric Caspase-9 Exhibits Higher Catalytic Activity and Stronger Cell-Killing Activity than the WT Protein
(A) A time course experiment of caspase-9 activity using procaspase-3 (C163A) as the substrate revealed that the dimeric caspase-9 (residues 1–416) exhibited an approximately 5-fold higher level of catalytic activity than the monomeric WT caspase-9. The concentrations were: WT and engineered caspase-9, 0.5 μM; procaspase-3 (C163A), 33 μM.
(B) Comparison of catalytic activity for the WT and dimeric caspase-9 using fluorescent substrate LEHD-AFC. Both the full-length and Δ139 dimeric caspase-9 displayed higher activities than their WT counterparts.
(C) The dimeric caspase-9 induced apoptosis more effectively than the WT protein. Mammalian expression vector pcDNA3 constructs encoding WT or dimeric caspase-9 were transfected into HeLa or 293 cells. The extent of cell death induced by each construct was examined.
(D) The dimeric but not the WT caspase-9 promoted the activation of caspase-3. The expression of caspase-9 variants (upper panel) and the processing of caspase-3 (lower panel) from the cell extracts were detected by antibodies against caspase-9 and -3, respectively. No endogenous caspase-9 band was visible in the vector-transfected cells at lower exposure.
Figure 5.
The Dimeric Caspase-9 Exhibits a Much Lower Activity Than the Apaf-1-Activated WT Caspase-9
(A) Using fluorescent substrate LEHD-AFC, the catalytic activity of the Apaf-1-activated WT caspase-9 was approximately 35-fold greater than that of the dimeric caspase-9.
(B) A time course comparison of the catalytic activities between the Apaf-1-activated WT caspase-9 and the dimeric caspase-9. WT and dimeric caspase-9, 0.5 μM; Apaf-1 (residues 1–570), 2 μM; LEHD-AFC, 200 μM.
Figure 6.
The Dimeric Caspase-9 Can No Longer Be Activated the Same Way as the WT Caspase-9
(A) Increasing amounts of Apaf-1 (residues 1–570) led to a corresponding increase in the catalytic activity of the WT caspase-9, but had no effect on the activity of the dimeric caspase-9. WT and dimeric caspase-9, 0.5 μM; procaspase-3 (C163A), 33 μM; Apaf-1, 0.24, 0.6, and 1.2 μM.
(B) A time course of procaspase-3 cleavage by the WT and dimeric caspase-9 in the presence of Apaf-1. WT and dimeric caspase-9, 0.3 μM; procaspase-3 (C163A), 33 μM; Apaf-1, 0.3 μM.