Lengthening the intersubunit linker of procaspase 3 leads to constitutive activation.
The conformational ensemble of procaspase 3, the primary executioner in apoptosis, contains two major forms, inactive and active, with the inactive state favored in the native ensemble. A region of the protein known as the intersubunit linker (IL) is cleaved during maturation, resulting in movement of the IL out of the dimer interface and subsequent active site formation (activation-by-cleavage mechanism). We examined two models for the role of the IL in maintaining the inactive conformer, an IL-extension model versus a hydrophobic cluster model, and we show that increasing the length of the IL by introducing 3-5 alanines results in constitutively active procaspases. Active site labeling and subsequent analyses by mass spectrometry show that the full-length zymogen is enzymatically active. We also show that minor populations of alternately cleaved procaspase result from processing at D169 when the normal cleavage site, D175, is unavailable. Importantly, the alternately cleaved proteins have little to no activity, but increased flexibility of the linker increases the exposure of D169. The data show that releasing the strain of the short IL, in and of itself, is not sufficient to populate the active conformer of the native ensemble. The IL must also allow for interactions that stabilize the active site, possibly from a combination of optimal length, flexibility in the IL, and specific contacts between the IL and interface. The results provide further evidence that substantial energy is required to shift the protein to the active conformer. As a result, the activation-by-cleavage mechanism dominates in the cell.
Department of Molecular and Structural Biochemistry and ‡Center for Comparative Medicine and Translational Research, North Carolina State University , Raleigh, North Carolina 27695, United States.
Sarah H
SH
MacKenzie
Joshua L
JL
Schipper
Erika J
EJ
England
Melvin E
ME
Thomas
Kevin
K
Blackburn
Paul
P
Swartz
A Clay
AC
Clark
D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology; D020134:Catalytic Domain; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D055503:Protein Multimerization
D061945:Caspase Inhibitors; D004792:Enzyme Precursors; D053148:Caspase 3