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Updated: Jun 21, 2025
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Crystal structure and mechanistic studies of the PPM1D serine/threonine phosphatase catalytic domain
Jay Prakash Kumar1, Dalibor Kosek2, Stewart R Durell1
1Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland, United States.
Abstract:
Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here, we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short β-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics.
Insights
We determined the first crystal structure of Protein phosphatase 1D (PPM1D), revealing its active site and unique structural features. This structure provides insights into PPM1D
Area of Science:
- Biochemistry
- Structural Biology
- Cancer Biology
Background:
- Protein phosphatase 1D (PPM1D, Wip1) is induced by p53 during DNA damage response.
- PPM1D functions as an oncoprotein in various human cancers.
- Its flexible regions have hindered structural determination, limiting therapeutic development.
Purpose of the Study:
- To determine the first crystal structure of the PPM1D catalytic domain.
- To investigate the structural basis for PPM1D's catalytic activity and regulation.
- To provide insights for developing PPM1D-targeted cancer therapeutics.
Main Methods:
- X-ray crystallography (1.8 Å resolution)
- Molecular dynamics simulations
- Enzyme kinetic studies
Main Results:
- The first crystal structure of the PPM1D catalytic domain was resolved, showing bound Mg2+ ions.
- Unique flap subdomain and B-loop structures crucial for substrate recognition were identified.
- A novel nitrogen-oxygen-sulfur bridge was discovered within the catalytic domain.
- Kinetic studies revealed magnesium-dependent hysteresis, indicating slow regulatory transitions.
Conclusions:
- The determined PPM1D structure advances understanding of its function and regulation.
- Structural insights, including the novel bridge and hysteresis, are key for therapeutic development.
- This work lays the foundation for designing PPM1D-specific inhibitors for cancer therapy.
