Lengthening the Intersubunit Linker of Procaspase 3 Leads to Constitutive Activation
Abstract
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.
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Cleavage of the intersubunit linker activates procaspase 3.
“Cleavage of the linker activates procaspase 3.”
From Protein Ensembles: How Does Nature Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell (2016) · cites this paper as [236]
The native conformational ensemble of inactive procaspase 3 is constrained by its intersubunit linker.
“The native conformational ensemble of inactive procaspase 3 is constrained by its intersubunit linker (IL).”
From Protein Ensembles: How Does Nature Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell (2016) · cites this paper as [236]
Releasing strain of the short intersubunit linker alone is insufficient to sufficiently increase the population of the active procaspase 3 conformer in the native ensemble.
“However, releasing the strain of the short IL is not enough to sufficiently increase the population of the active conformer in the native ensemble.”
From Protein Ensembles: How Does Nature Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell (2016) · cites this paper as [236]
Increasing the intersubunit linker length by introducing 3-5 alanines can produce constitutively active procaspases.
“Interestingly, increasing the IL length by introducing 3–5 alanines can lead to constitutively active procaspases.236”
From Protein Ensembles: How Does Nature Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell (2016) · cites this paper as [236]
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Wei, G., Xi, W., Nussinov, R., & Ma, B. (2016) · Chemical Reviews · doi:10.1021/acs.chemrev.5b00562 · Cited by 436 · see citing works
- Authors
- MacKenzie, S. H., Schipper, J. L., England, E. J., Thomas, M. E., Blackburn, K., Swartz, P., & Clark, A. C.
- Affiliations
- North Carolina State University (US)
- Corresponding author
- Clark, A. Clay
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2013-08-27
- Volume
- 52
- Issue
- 36
- Pages
- 6219-6231
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2316933980
- Cited by
- 15
- References
- 43
- Citations by year
- 2025: 1 · 2024: 1 · 2023: 1 · 2021: 1 · 2020: 1 · 2017: 2 · 2016: 4 · 2015: 1 · 2014: 3
- FWCI
- 1.19
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM 065970, R01GM065970)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
- Keywords
- procaspase-3, constitutive activation, active site exposure, hydrophobic clusters, protein flexibility, apoptosis
- MeSH terms
- Enzyme Activation, Enzyme Precursors, Models, Chemical, Models, Molecular, Protein Conformation, Apoptosis, Catalytic Domain, Caspase 3, Protein Multimerization, Molecular Dynamics Simulation, Caspase Inhibitors
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