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Correlation of structure and function in the human hotdog-fold enzyme hTHEM4
Hong Zhao1, Kap Lim, Anthony Choudry
1Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131, USA.
Biochemistry
|August 9, 2012
Summary
Human THEM4 (hTHEM4), a protein linked to cell apoptosis, directly binds and inhibits Akt1 kinase activity. This study reveals the X-ray structure of hTHEM4 and confirms its role as an Akt1 negative regulator.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Human THEM4 (hTHEM4) possesses a catalytic acyl-CoA thioesterase domain and an N-terminal domain of unknown function.
- hTHEM4 has been implicated in the regulation of Akt1 and cellular apoptosis.
Purpose of the Study:
- To elucidate the structure of hTHEM4 bound to undecan-2-one-CoA.
- To investigate the functional interaction between hTHEM4 and Akt1.
- To confirm the catalytic residues of hTHEM4 through structure-guided mutagenesis.
Main Methods:
- X-ray crystallography to determine the structure of hTHEM4-undecan-2-one-CoA complex.
- Structure-guided site-directed mutagenesis to identify key catalytic residues.
- Immunoprecipitation assays to demonstrate direct binding between hTHEM4 and Akt1.
- Enzyme activity assays to assess the effect of hTHEM4 on Akt1 kinase activity.
Main Results:
- The X-ray structure of hTHEM4 bound to undecan-2-one-CoA was determined.
- Mutagenesis confirmed the catalytic residues essential for hTHEM4 function.
- The N-terminal domain of hTHEM4 exhibits irregular and flexible secondary structures, suggesting a protein-binding role.
- Direct binding between hTHEM4 and Akt1 was confirmed.
- hTHEM4 was shown to inhibit Akt1 kinase activity.
Conclusions:
- hTHEM4 directly binds to Akt1.
- hTHEM4 functions as a negative regulator of Akt1 kinase activity.
- The structural and biochemical data provide a foundation for understanding hTHEM4's role in cellular processes.
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